Progress 10/01/09 to 09/30/13
Outputs
OUTPUTS: This project was initiated in 1978 and the main focus has been to study the regulation of motility in a variety of tissues and cells. The early work centered on smooth muscle and here it was found that the major mechanism for regulation of contractile activity was based on phosphorylation of myosin, specifically the two 20kD light chains,LC20. Phosphorylation induced contraction and dephosphorylation favored relaxation. The major components for this mechanism are a myosin light chain kinase (MLCK)and a myosin phosphatase (MP). A critical feature was the discovery that MLCK is activated by the Ca-binding protein ,calmodulin (CaM). Thus the increase of intracellular Ca,that induces contraction (in all muscles),forms the Ca-CaM complex that activates MLCK, promotes phosphorylation of myosin and leads to contraction.This basic process is proposed to be involved in initiation of motile processes in many cells, not just in smooth muscle.(Note that the initiation of contraction in striated muscle is regulated by the troponin- tropomyosin complex). Elucidation of the molecular details for MP proved more complex.But it was later shown that MP is composed of 3 subunits:a PP1c catalytic subunit, delta isoform,a large (~110kD)subunit,termed the myosin phosphatase target subunit, MYPT; and a smaller subunit of 20kD, M20 of unknown function> Most of the properties of MP can be replicated by an equistoichiometric complex of PP1c and MYPT.MYPT was cloned (sequenced) and many of its critical features were established.The basic mechanism for MP is as follows: PP1c binds to a specific sequence of 4 residues at the N-terminus of MYPT, the PP1c target site. The LC20s bind next to the PP1c site of MYPT. The myosin rod is thought to interact with a coiled-coil region of MYPT, close to the C-terminus. The myosin heavy chain is approximately 200kD and thus interaction of the LC20s at the myosin head at the N-terminus of MYPT and binding of the myosin rod at the C-terminus of MYPT is feasible.Perhaps the most critical discovery was that MYPT is the regulatory component of MP and that regulation hinges on phosphorylation MYPT.The phosphorylation sites are T695 and T850 (for the gizzard isoform) and phosphorylation of either or both inhibits MP activity and increases the level of myosin phosphorylation, favoring contraction (the so-called Ca-sensitization effect).Activation of MP and resultant relaxation of smooth muscle (the "off" signal)is induced by an increased concentration of cyclic nucleotides (cAMP and cGMP).Although the molecular details for this mechanism are not established it is thought to involve interactions at the C-terminus of MYPT. Again it should be emphasized that these basic mechanisms operate both in smooth muscle and many motile cells.Later it was found that a different isoform of MYPT (termed MYPT2)was found in skeletal and cardiac muscle and although the structures of MYPT1 and MYPT2 are similar there are critical differences,mostly at the N-terminal ends. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The obvious and initial impact was the realization that a similar mechanism could account for regulation of motility in smooth muscle and non-muscle cells and the previously held notion that skeletal muscle is the prototype for motile systems is incorrect.One potential application is that chemotaxis of cancer cells is due to the MLCK-MP system.Another critical aspect was discovered more gradually but was initiated by the cloning of MYPT1. It was found that MYPT contained several different protein interaction sites: towards the N-terminus there are many ankyrin repeats and these are established protein-protein interaction sites;also the phosphorylation sites T695 and T850 obviously bind kinases; and close to the C-terminus is an extensive coiled-coil region,again established interaction sites. The presence of these regions (and probably others)raises the possibility that MYPT1 and 2 can form complexes with several partners and indeed this realization has been demonstrated. It was discovered that MYPT binds to: cytoskeletal proteins ezrin,radixin and and moesin (ERM proteins)and thus could be involved in regulated/organization of the cytoskeleton;Tau and MAP2 (tubulin regulation);HDAC7 (epigenetics); components of the cGMP pathway; and components of the nucleus (see Hartshorne and Matsumara, Bioch. Biophys.Res. Commun.369,149[2008]) The latter is critical and we have shown that MYPT1 is important in the regulation of mitosis. MYPT1 is transported into the nucleus via an N-terminal nuclear localization sequence and is effective in the G1/S phase where it regulates phosphorylation of the checkpoint protein, retinoblastoma protein (Rb).Rb binds to the C-terminal part of MYPT1. Later in M phase. MYPT1 is phosphorylated by Cdc2 to form a inding site for polo-like kinase (PLK1). The activity of PLK1 is regulated by phosphorylation and its interaction with MYPT1 leads to its dephosphorylation.Finally,at the end of M phase in cytokinesis the phosphorylation of myosin 2 is controlled by the MYPT1-PP1c complex.Many kinases phosphorylate MYPT1,notably Rho-kinase (ROK). Phosphorylation of MYPT1 (predominantly at T850)by ROK inhibits MP activity and in smooth muscle this increases contraction and this has been suggested as a component of hypertension.Several ROK inhibitors have been developed and use clinically to treat hypertension.The role(s) of many of the kinases that phosphorylate MYPT1 (eg.integrin-linked kinase; myotonic dystrophy kinase)has not been established.Over the last few years there have been many studies on MYPT1 but few on MYPT2.Recently,this has been shown to be important in cardiac mechanics and ultrastructure. A transgenic mouse was developed in which MYPT2 was over-expressed (by ~6 fold). Unexpectedly this also led to an increase in PP1c expression and thus cardiac MP activity was increased.The higher MP activity led to:a decrease in Ca-sensitivity;an increase in end-systolic and end-diastolic volumes and reduced ejection fraction;hypertrophy of the LV; and both systolic and diastolic functions were impared. These findings (and others)emphasize the critical role of myosin phosphorylation (and hence MLCK and MP) in cardiac function.
Publications
- No publications reported this period
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: Myosin phosphorylation at the 2 regulatory light chains is recognized as an important mechanism controlling a variety of motile events, including: smooth muscle contractile cycle (the first mechanism discovered); potentiation of contraction in skeletal and cardiac muscle; cell motility, including metastasis of cancer and other cells; and cytokinesis. All of these processes require myosin II and its phosphorylation level is controlled by myosin light chain kinase (MLCK) and a myosin phosphatase (MP). MLCK is regulated by Ca-calmodulin and thus its activity tracks the intracellular Ca levels. MP is composed of 3 subunits: the catalytic subunit, PP1c; a targeting subunit, MP target subunit (MYPT); and a smaller subunit of unknown function (M20). Regulation of MP is complex and only recently discovered. MYPT is the regulatory subunit and several regulatory mechanisms, or schemes, have been proposed. These reflect the varied domain structure of MYPT: an N-terminal PP1c binding motif; an N-terminal series of ankyrin repeats; an acidic region; a S/T rich sequence; and C-terminal coiled-coil domains. Plus there are several phosphorylation sites for many kinases, both activating and inhibitory. Thus MYPT should be regarded as an adaptor or platform protein that via its wealth of different domains can interact and regulate several proteins, not just myosin II. Also the intracellular location of MYPT/PP1c changes and is subject to different signaling cascades. It binds to the plasmalemma and interacts with several proteins, eg ezrin, moesin and radixin. It may also be localized to caveolae and/or cell junctions (for example in confluent cells the extent of membrane attachment of MYPT is markedly increased).With the cytoskeletal proteins it controls the stability of stress fibers (in cultured cells) via myosin II phosphorylation and is involved with microtubules, Tau and MAP2. A surprising finding was that it also enters the nucleus and interacts with merlin (a tumor suppressor), HDAC 7 (a transcriptional repressor that has both cytosolic and nuclear locations) and regulates cell cycle progression via retinoblastoma (Rb - G1/S checkpoint) and regulates polo-like kinase 1 (Plk-1) at the metaphase-anaphase transition. For the nuclear functions it is unlikely that myosin II is a substrate until the nuclear membrane dissolves at prometaphase and it is likely that several nuclear partners remain to be identified. This diversity in function for MP was an exciting discovery and opening many avenues for research and the possibility of different signaling pathways. Initially 2 pathways were suggested: an inhibitory pathway involving RhoA/Rho kinase or ZIPK and resulted in phosphorylation of the inhibitory sites; and an activating pathway, implicating cyclic nucleotides, cAMP and/or cGMP. Now we can add cyclin-dependent kinases and phosphorylation of the "cell - division" sites and possibly PKC (with proposed function in nuclear translocation).But molecular mechanisms and details on translocation and target proteins are still lacking and are obviously priorities for future research. The discovery of different MYPT isoforms has added to the complexity. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
One way to investigate the many roles of MYPT is to identify interacting proteins. We have used several approaches to address this issue: One approach was to use the yeast 2 -hybrid system. This was an extension of our earlier work, but varied by using MYPT as bait to screen an aorta library. Several positives were obtained but 2 were of particular interest, ie CD81, a 26kD cell surface molecule involved in many signaling pathways and phosphofructokinase (PFK - muscle isoform, PFK-M) the rate limiting step in glycolysis and known to be regulated by phosphorylation. We focused more on PFK-M and found that MYPT did bind to PFK-M using Elisa assays, but effects on regulation remain to be established. Also PFK-M was detected in IPs using MYPT antibodies. (The antibodies to PFK-M were adequate to detect PFK-M but could not be used for IP). It is interesting that in skeletal muscle, PFK-M has been shown to bind to thin filaments (in addition to several other enzymes of glycolysis) and in smooth muscle is thought to be localized (at least in part) at caveolae; It is accepted that MYPT binds myosin and one theory for regulation is that phosphorylation of MYPT by ROK or ZIP disrupts myosin binding. This has been documented using pull-down assays (our laboratory) but although this may be important in stress fiber stability it does not fully rationalize the regulatory mechanism since the dephosphorylation of P- light chains is also regulated and these are not dissociated on phosphorylation of MYPT. The examine some of these binding parameters we used Surface Plasmon Resonance (SPR) and the results are summarized below: a) Binding of light chain (LC20) to MYPT. LC20 binds with relatively high affinity (KD in μM range) to full-length MYPT and the N-terminal fragment (1-296) but not to the C-terminal fragment (667-1004). b) There was no major difference in binding of LC20 and P-LC20 to both full-length MYPT and the N-terminal fragment, although there was a slight preference for P-LC20. (In gel overlay experiments there was a preference for P-LC20 and this probably reflected the markedly different conditions). c) The C-terminal fragment binds to full-length MYPT and 1-296 (with approximately equal affinity), but forms dimers with much lower affinity (arguing for interaction between the C- and N-terminal ends of MYPT). d) Binding to myosin indicated preferential binding of the C-terminal and full-length MYPT but less with 1-296. This probably reflects the presence of the C-terminal myosin-binding site and the reduced binding to 1-296 the binding of the myosin light chain. In general these assays were confirmed using ELISA, although these were more difficult to quantitate. Additional points from ELISA were that P-myosin binds stronger to both the N- and C-terminal fragments of MYPT and that the addition of ATP to the binding media increases binding affinity. Adding to the complexity of the situation is the presence of several isoforms of MYPT, notably MYPT2 found in striated muscle. In cardiac muscle we showed by overexpressing MYPT2 that it functions to maintain structural and functional integrity of cardiac performance .
Publications
- No publications reported this period
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: Phosphorylation of myosin II is important in many aspects of cell function and the diversity of mechanisms in which this process is involved is increasing rapidly. The mechanism utilizes a myosin ligh chain kinase (MLCK) and a myosin phosphatase (MP) and these operate in opposite directions. MLCK is dependent on Ca via interaction with calmodulin and MP has several potential regulatory mechanisms. Our recent studies have focused on MP and this was shown to be composed of 3 subunits: a catalytic subunit, PP1cδ; a targeting subunit, myosin phosphatase tartget subunit (MYPT); and a small subunit (M20). All of the known functions of MP can be reproduced using only PP1c and MYPT. A feature of MYPT is its ability to interact with several distinct molecules utilizing different regions of the molecule. Myosin binds to the N-terminal end, close to and possibly involving the multiple ankyrin repeats. Ankyrin repeats are known to provide a platform for protein interaction and thus this finding was anticipated. However, subsequent findings indicated that the C-terminal end of MYPT also was important in binding to many other proteins. This was an exciting discovery and indicated a much greater flexibility for MP function than originally envisaged. Some of the proteins linked to MP function: the membrane proteins, ezrin, radixin and moesin (ERM complex); tau and MAP2; Rho-A - GTP; PKG; merlin (a tumor suppressor); HDAC 7 (a transcriptional repressor); and retinoblastoma protein (Rb). [These interactions are described in Matsumura and Hartshorne, Biochem. Biophys. Res. Commun. 369,149,2008]. Our interests have been directed toward understanding details of several of these interactions (ie, which parts of the respective molecules are involved) and how is the interaction regulated to conform with cell function. A major regulatory factor is phosphorylation of MYPT at different sites by various kinases. We have shown that Rho-kinase, zipper-interacting kinase (ZIPK), and cdk1 phosphorylate MYPT at different sites. These in general are grouped into the inhibitory sites (for Rho-kinase and ZIPK) and the "cell division" sites( for cdk1) and each controls a different aspect of cell function. It is known from several analytical studies that MYPT is located both in the cytosol and in the nucleus. It contains 2 separate nuclear localization signals, 1 in the N-terminus and 1 at the C-terminus. Our recent focus has been on the role(s) of MYPT in nuclear function and with respect to the regulatory phosphorylation sites both regions are involved. Thus we have found both inhibition of MP activity and modification of MYPT interactions with proteins associated with cell cycle. Several phases of cell cycle are implicated. In the early stages of the cell cycle, G1, S, and G2 the nuclear membrane is still intact and thus a nuclear translocation mechanism must operate and this topic is the subject for future research, particularly whether phosphorylation at a T residue close to the N-terminal NLS by PKC regulates translocation. Later in M phase the membrane dissolves. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Our first study on nuclear function of MYPT used a mammalian expression vector designed for several GFP-tagged constructs to over-express MYPT1 in NIH 3T3 and HeLa cells. Only those constructs containing the N-terminal NLS entered the nucleus. The full-length constructs showed localization in discrete nuclear foci. The major effect of over-expression was a decrease in the number of attached cells and an apparent block in cell-cycle progression before M phase plus signs of increased apoptosis. An interesting observation was that under serum-starved conditions the translocation of MYPT into the nucleus was blocked and there was no decrease in the number of attached cells. With viral - transformed cells (HeLa) stable transfection was obtained. The MYPT formed a complex with Rb protein and this was the key to the first part of these studies. The conclusions were that the binding of MYPT to Rb facilitates dephosphorylation of Rb and thus blocks progression of the cell cycle from G1 to S phase. With viral transformed cells, viral proteins act to bypass this block, ie, they act as alternative transcription factors. The effect of serum starvation is an area for future research. Our working hypothesis is that in the absence of serum the Ras/Raf pathway is not activated and PKC also is inactive. Thus it is suggested that phosphorylation of MYPT near the N-terminal NLS by PKC is necessary for nuclear translocation and effects on G1/S transition. MYPT also acts at the end of the cell cycle, in cytokinesis. It was found that phosphorylation of myosin II activates the primary motor for cell separation and thus dephosphorylation should be minimized. This is achieved by phosphorylation of MYPT by Rho-kinase at the inhibitory sites. There are questions remaining: first to what extent is MP critical for cytokinesis and focuses on which kinases might also be involved and whether these can direcly phosphorylate myosin II. Candidates are MLCK, Rho-kinase and citron kinase. Second; which signaling pathways are responsible for inhibition of MP. The favored pathway is the RhoA-Rho-kinase link but other signaling events could contribute. In addition to its role in cytokinesis MP appears to be implicated at an earlier part of M phase. It was shown in recent studies (in collaboration with F.Matsumura) that MP antagonizes polo-like kinase 1 (PLK1). Cdk1 phosphorylates MYPT and this promotes binding of PLK1. The ensuing antagonism prevents precocious chromatid separation before the onset of anaphase. A suggested mechanism is that the binding of PLK1 to MYPT blocks phosphorylation by PLK1 of a cohesion subunit, SA2, at the centromere. Several aims have been proposed for ongoing research to test this hypothesis. One hypothesis is that binding of PLK1 to MP promotes dephosphorylation of PLK1 at its activating site, T210. Thus securing chromosome cohesion at the centromeres before the onset of anaphase, acting as a cycle check - point. A second hypothesis is that MP/ MYPT share common substrates /binding partners and thus competition would limit or restrict phosphorylation of PLK1 at its activating site. Several aims are proposed to check these 2 hypotheses.
Publications
- Mizutani, H., Okamoto, R., Moriki, N., Konishi, K., Taniguchi, M., Fujita, S., Dohi, K., Onishi, K., Suzuki, N., Satoh, S., Makino, N., Itoh, T., Hartshorne, D., Ito, M., (2010). Overexpression of Myosin Phosphatase Reduces of Ca2+ sensitivity of Contraction and Impairs Cardiac Function. Circ. j. 74, 120-128
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: The phosphorylation scheme for activation of myosin and actin-activated ATPase activity was established from studies carried out with smooth muscle. Initially chicken gizzard was used as a convenient source, but subsequently several smooth muscles were analyzed. The basic components of the scheme are that the regulatory light chains of myosin (LC20) are phosphorylated by a Ca-calmodulin -dependent myosin light chain kinase (MLCK) and this activates the contractile apparatus. The "off" switch is dephosphorylation by a myosin phosphatase (MP). Initially it was thought that MP was not regulated but when MP was characterized it was found that both activation and inhibition of MP occurred. MP is composed of 3 subunits: a PP1 catalytic subunit, δ isoform (PP1cδ); a large regulatory subunit, termed myosin phosphatase target subunit (MYPT); and a small subunit, M20, of unknown function. Most of the properties of MP can be replicated by the complex of PP1cδ and MYPT and regulation of MP is governed by MYPT. Later it was found that there are 2 genes for MYPT, expressing MYPT1, found in most cells but high in smooth muscle: and MYPT2, found predominantly in brain and striated muscle. Both molecules have similar structures and it was assumed that each was dedicated to dephosphorylation of the respective myosins. A major function for each isoform is regulation of myosin, but for MYPT1 it was discovered to be involved in a wide range of cell functions and these reflect binding of different substrates to the MYPT1 molecule. One area in which our laboratory has focused is the role of MYPT1 in the nucleus. There are several phases of the mitotic cycle in which MYPT1 is involved. It is transported into the nucleus via an N-terminal nuclear localization sequence and is effective in the G1/S phase where it regulates phosphorylation of the check point retinoblastoma protein (Rb). Rb binds to the C-terminal part of MYPT1. Later in M phase, MYPT1 is phosphorylated by Cdc2 to form a binding site for polo-like kinase (PLK1). The activity of PLK1 is regulated by phosphorylation and its interaction with MYPT1 leads to its dephosphorylation. Finally at the end of M phase in cytokinesis the phosphorylation of myosin II is regulated by MYPT1. Other cell functions for MYPT1 involve regulation: of the cytoskeletal proteins, ezrin, radixin and moesin; Tau and MAP2; merlin, a tumor suppressor protein; HDAC 7; and components of the cGMP pathway ( summarized in Hartshorne and Matsumura, Biochem. Biophys. Res. Commun.369, 149 [2008]). PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Over the last few years there have been several studies on MYPT1 but few on MYPT2. It was established from genomic sequence that its structure is similar to MYPT1 and differs only in the N-terminal region. This region is important in activation of PP1cδ and binding to the light chain substrate and it was therefore suggested that the difference in N-terminal sequences reflected targeting to different myosin isoforms. One tissue in which MYPT2 is expressed is cardiac muscle and to evaluate its role in cardiac function, and specifically in dephosphorylation of cardiac myosin, a transgenic mouse was developed in which MYPT2 was over-expressed. The heart-specific Transgenic mouse (Tg) was generated using the α - myosin heavy chain (MHC) promoter and this model was analyzed at the molecular, cellular and whole animal levels. The level of overexpression of MYPT2 was approximately 6 fold higher than in wild type (wt) mice. An unexpected finding was that in the Tg mice the level of PP1cδ also was increased at about an equimolar ratio to MYPT2. Thus effectively raising the level of the MP holoenzyme and increasing type 1 phosphatase activity. The level of the M20 subunit was not changed in the Tg mice. The increased MP activity was reflected by a lower phosphorylation level of LC-20 and in permeabilized fibers a slight decrease in Ca sensitivity. Several of the hemodynamic parameters were altered in the Tg mice, including: an increase in end - systolic and end - diastolic volumes and a reduced ejection fraction compared to wt; a slight reduction in cardiac index and stroke volume index; an overall decrease in left ventricular contractility and a prolonged LV relaxation rate. These changes suggested that the LV was enlarged and both systolic and diastolic function were impaired. Other hypertrophy markers, brain natriuretic peptide and β- MHC (but not α- MHC), also were increased in Tg mice. There were no marked histological or ultrastructural changes. These results indicate the importance of myosin phosphorylation in long - term cardiac performance and emphasises the critical role that regulation of myosin phosphorylation levels plays in cardiac function. In the latter respect MP and MYPT2 are essential components. It remains to be determined if MYPT2 has a wide range of functions in cardiac muscle that MYPT1 has in many other cell types. An interesting finding made during the study on Tg hearts was that in the absence of Ca there was still significant phosphorylation of LC20, indicating either a Ca-independent form of MLCK or the novel concept that another kinase can phosphorylate cardiac myosin.
Publications
- Funabara D, Osawa R, Ueda M, Kanoh S, Hartshorne DJ, Watabe S. (2009). Myosin loop 2 is involved in the formation of a trimeric complex of twitchin, actin, and myosin. J Biol Chem. 284, 18015-20. Mizutani, H., Okamoto, R., Moriki, N., Konishi, K., Taniguchi, M., Fujita, S., Dohi, K., Onishi, K., Suzuki, N., Satoh, S., Makino, N., Itoh, T.,, Hartshorne, D., Ito, M., (2010). Overexpression of Myosin Phosphatase Reduces of Ca2+ sensitivity of Contraction and Impairs Cardiac Function. Circ. j. 74, 120-128
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: Myosin phosphatase (MP) was isolated and characterized first from smooth muscle and in this tissue its main role is to regulate the level of myosin phosphorylation and thus the contractile activity of the muscle. The nomenclature derived from the fact that phosphorylated myosin (P-myosin) was the substrate and also led to the term used for its major regulatory subunit, myosin phosphatase target subunit (MYPT). MYPT acts to regulate the catalytic subunit (PP1cδ) and also colocalizes the holoenzyme to various substates and subcellular locations. It is now known that there are several substrates for MP and that MP is involved in many cell processes, not just in the smooth muscle contraction-relaxation cycle. These are summarized in Matsumura and Hartshorne (2008). MP is found in all cell fractions and is translocated to different locations by varying the mode of cell stimulation, ie. by distinct signal transduction pathways. The details remain to be established. One organelle in which MP plays critical roles is the nucleus. Earlier work from our laboratory showed that MYPT was translocated to the nucleus and blocked the G1/S transition, possibly by dephosphorylation of retinoblastoma protein. We have now identified a second site of action, namely during M phase (Yamashiro et al. 2008). MYPT1 is phosphorylated by the major mitotic cyclin-dependent kinase, Cdc2, and this forms a binding site for polo-like kinase (PLK1). PLK1 is activated by phosphorylation and its interaction with MYPT promotes dephosphorylation at Thr 210 and inactivation of PLK1. Thus MP and PLK1 have antagonizing roles and by this means MP plays a critical role in progression of the mitotic cycle. The site(s) on MYPT phosphorylated by Cdc2 include Ser 472, but other sites may be involved and we are identifying these. If other sites are identified the roles of individual sites will be assessed. An interesting feature of the nuclear translocation of MYPT is that it does not occur in serum-starved cells and thus the signal for translocation is associated with a growth factor-induced signaling pathway. The N-terminal nuclear localization signal (NLS) is adjacent to a PKC site and thus phosphorylation of MYPT by PKC may activate translocation into the nucleus. There are several gene families for MYPT-like proteins. The 2 families that we focus on are MYPT1 and MYPT2. The former is ubiquitous and has several splicing isoforms. The latter is found predominantly in striated muscle and brain. We have overexpressed MYPT2 in cardiac muscle in a mouse model. Increased expression of MYPT2 was accompanied by a proportionate increase in the PP1 catalytic subunit, thus the level of MP increased. This was reflected by a decreased level of myosin phosphorylation in skinned cardiac fibers and a slight shift in Ca2+- sensitivity of contraction. Over the long term the decrease in myosin phosphorylation induced cardiac hypertrophy, LV enlargement and impaired cardiac function. These results indicate that regulation of myosin phosphorylation in the heart is critical to maintain normal cardiac function. It is interesting that an increase in myosin phosphorylation enhances cardiac function. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The new concept that emerges from these studies is that MP and its major subunit, MYPT1, has a much broader role in cell function than regulation of myosin II-dependent motile processes, as thought previously. This changes the direction of future studies and places an onus on understanding the interactions of MYPT1 with multiple partners. Some of the interacting proteins and associated cell processes are: Myosin II, motor functions and cytoskeletal organization; ezrin, radixin and moesin (ERM), cytoskeletal organization; α-adducin, actin membrane attachment; Tau and MAP2, microtubule dynamics; ZIP kinase, phosphorylation of MYPT1 and possibly myosin II; PKG, cGMP-dependent relaxation of smooth muscle; interleukin-16 precursor, cytokine precursor; merlin, a tumor suppressor protein; HDAC 7, transcriptional repressor; HSP 27, RhoA-docking protein; and synaptophysin, neurotransmitter release in synaptic vesicles. The siRNA results indicate that other cell functions also are affected by MYPT1 knockdown, eg. cell adhesion and microfilament stability. MYPT1 is involved in The ability of MYPT1 (and possibly MYPT2) to bind many proteins depends on both the N-terminal ankyrin repeats and the C-terminal region (where an important segment is the coiled-coil sequence). Using a variety of truncation mutants representing different parts of the MYPT1 molecule we are mapping interaction sites for several of the putative binding partners. In addition, we have initiated the yeast 2-hybrid screen to probe for interactions in smooth muscle (aorta). There are several splicing isoforms of MYPT1 that affect the central region and the C-terminal leucine zipper (LZ) repeats. The individual roles of these isoforms is not established, but it is suggested that the LZ repeats are implicated in PKG binding and the expression of certain isoforms varies on conditions. (An example of this is that one of the 3 isoforms found in A7r5 cells is found only at confluence). The challenge for our laboratory and several other groups is to define the role of individual isoforms and to determine if their translocation and location in the cell are distinct For example we are investigating whether specific isoforms are involved in the cell response to cyclic nucleotides and whether distinct isoforms are translocated into the nucleus The present studies are focused on MYPT1 and there are no corresponding data with MYPT2.In future studies it should be established whether there are isoforms of MYPT2 and whether the PP1c/ MYPT2 complex has additional substrates (in addition to P-myosin). These data would help establish the role(s) of MYPT2 in cardiac and brain function.
Publications
- Matsumura, F., and Hartshorne, DJ. (2008). Myosin phosphatase target subunit: Many roles in cell function. Biochem Biophys Res Commun. 369: 149-156.
- Yamashiro, S., Yamakita, Y., Totsukawa, G., Goto, H., Kaibuchi K., Ito M., Hartshorne, DJ., Matsumura, F.(2008). Myosin phosphatase-targeting subunit 1 regulates mitosis by antagonizing polo-like kinase 1. Devel. Cell 14: 787-797.
- Cremo, C.R., and Hartshorne, D.J. (2008). Smooth-Muscle Myosin II in "Myosin: A Superfamily of Molecular Motors"(ed. L. M. Coluccio) pp171-222, Springer 2008
- Kusaka, M., Ikeda, D., Funabara, D., Hartshorne, DJ. and Watabe, S.(2008). The occurrence of tissue-specific twitchin isoforms in the mussel Mytilus galloprovincialis. Fish. Sci. 74: 677-686
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Progress 01/01/07 to 12/31/07
Outputs
Several studies indicate that the major regulatory subunit of myosin phosphatase, ie the myosin phosphatase target subunit (MYPT1) is involved in many cell functions, not just the dephosphorylation of myosin II. The MYPT1 subunit acts to colocalize the catalytic subunit of phosphatase type 1 (PP1c) to various substrates. An important and previously unrecognized example is the binding of MYPT1 to polo-like kinase (PLK1) during mitosis. MYPT1 is phosphorylated during mitosis by cdc2 and this generates a binding motif for the polo box domain of PLK2. Depletion of PLK2 by siRNA results in loss of gamma-tubulin recruitment to the centrosomes and leads to mitotic arrest. Co-depletion of MYPT1 and PLK2 reinstates gamma-tubulin at the centrosomes and rescues mitotic arrest. MYPT1 depletion increases phosphorylation of PLK1 at the activating site (Thr210) and this in part rationalizes the rescue phenotype by co-depletion. The reduction in MYPT1 also induces cell detachment and
cell death (anoikis). Those cells that survive and remain attached are frequently multi-nucleate. This suggests that MYPT1 also plays a role in cytokinesis. The latter reflects the dephosphorylation of myosin II at the cleavage furrow. It is known that PP1c is important in various nuclear functions and is implicated at various phases of the mitotic cycle. Previously it was shown in studies from our laboratory that over-expression of MYPT1 and various subfragments were localized to the nucleus and blocked the G1/S transition. This probably reflects dephosphorylation of the retinoblastoma protein, thus ablating its check-point function. Binding of the retinoblastoma protein was found to occur with the C-terminal half of MYPT1. An intriguing aspect that will be investigated in future studies, was the finding that in serum-starved cells the MYPT1 constructs were not targeted to nuclei but remained cytoplasmic. This suggests that signaling via growth factor receptors determines cell
location of MYPT1 and the hypothesis is that protein kinase C is involved, possibly by phosphorylation at a site close to the N-terminal nuclear localization sequence.
Impacts
An important contribution of the above studies is that it supports the emerging theme that MYPT1 is involved in many aspects of cell function and contrary to the earlier view is involved in dephosphorylation of many substrates. Myosin phosphatase was characterized initially from smooth muscle (gizzard) and it was assumed that it was specific for myosin II in the contractile apparatus. Later work established that myosin phosphatase was wide spread and found in many cell types and had a much more diverse function than just dephosphorylation of myosin II.Within the nucleus MYPT1 has at least two partners, ie. retinoblastoma protein and PLK1. The results obtained with PLK1 identify a previously unrecognized role for MLK1 in regulating mitosis by antagonizing PLK1.After prometaphase when the nuclear membrane is disrupted, myosin II would become accessible to MYPT1and is involved in cytokinesis. (It is thought that myosin II is not present in membranated nuclei). The ability
of MYPT1 (and possibly its isoform, MYPT2) to bind to many different substrates reflects interactions with the N-terminal ankyrin repeats and the C-terminal region, including the coiled-coil sequences. Thus an exciting feature of these studies is the emerging concept that MYPT1 may be a scaffolding protein and is utilized at different cell locations to facilitate varied cell functions. The siRNA results indicate that several other cell functions are influenced by MYPT1 knock-down, notably cell adhesion and microfilament stability, and the role of MYPT1 in these processes must be determined.
Publications
- Funabara D, Hamamoto C, Yamamoto K, Inoue A, Ueda M, Osawa R, Kanoh S, Hartshorne DJ, Suzuki S, Watabe S. 2007. Unphosphorylated twitchin forms a complex with actin and myosin that may contribute to tension maintenance in catch. J Exp Biol 210:4399-4410.
- Matsumura F. and Hartshorne DJ. 2008. Myosin phosphatase target subunit: Many roles in cell funtion. Biochem.Biophys.Res.Commun. in press
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Progress 01/01/06 to 12/31/06
Outputs
Urocortin, a vasodilatory peptide related to corticotropin-releasing factor, may be an endogenous regulator of blood pressure. In vitro, rat tail arteries are relaxed by urocortin by a cAMP-mediated decrease in myofilament Ca2+ sensitivity through a still unclear mechanism. It is shown that contraction of intact mouse tail arteries induced with 42 mmol/L KCl or 0.5 micromol/L noradrenaline was associated with a approximately 2-fold increase in the phosphorylation of the regulatory subunit of myosin phosphatase (SMPP-1M), MYPT1, at Thr696, which was reversed in arteries relaxed with urocortin. Submaximally (pCa 6.1) contracted mouse tail arteries permeabilized with alpha-toxin were relaxed with urocortin by 39+/-3% at constant [Ca2+], which was associated with a decrease in myosin light chain (MLC20Ser19), MYPT1Thr696, and MYPT1Thr850 phosphorylation by 60%, 28%, and 52%, respectively. The Rho-associated kinase (ROK) inhibitor Y-27632 decreased MYPT1 phosphorylation by
a similar extent. Inhibition of PP-2A with 3 nmol/L okadaic acid had no effect on MYPT1 phosphorylation, whereas inhibition of PP-1 with 3 micromol/L okadaic acid prevented dephosphorylation. Urocortin increased the rate of dephosphorylation of MLC20Ser19 approximately 2.2-fold but had no effect on the rate of contraction under conditions of, respectively, inhibited kinase and phosphatase activities. The effect of urocortin on MLC20Ser19 and MYPT1 phosphorylation was blocked by Rp-8-CPT-cAMPS and mimicked by Sp-5,6-DCl-cBIMPS. In summary, these results provide evidence that Ca(2+)-independent relaxation by urocortin can be attributed to a cAMP-mediated increased activity of SMPP-1M which at least in part is attributable to a decrease in the inhibitory phosphorylation of MYPT1.
Impacts
The impact of myosin phosphatase is a critical component of smooth muscle function. Modification of normal phosphatase activity leads to several smooth muscle disorders, including hypertension. cAMP is an important component in regulation of myosin phosphatase.
Publications
- Lubomirov, L.T., Reimann, K, Metzler, D., Hasse, V., Stehle, R., Ito, M., Hartshorne, D.J., Gagov, H., Pfitzer, G. and Schubert, R. (2006). Urocortin-Induced Decrease in Ca2+ Sensitivity of Contraction in Mouse Arteries Is Attributable to cAMP-Dependent Dephosphorylation of MYPT1 and Activation of Myosin Light Chain Phosphatase. Circ. Res. 98, 1159-1167
- Funabara D, Kanoh S, Siegman MJ, Butler TM, Hartshorne DJ, Watabe S. (2005).Twitchin as a regulator of catch contraction in molluscan smooth muscle. J Muscle Res Cell Motil. 26(6-8):455-60
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Progress 01/01/05 to 12/31/05
Outputs
Major sites for Rho-kinase on the myosin phosphatase target subunit (MYPT1) are Thr695 and Thr850. Phosphorylation of Thr695 inhibits phosphatase activity but the role of phosphorylation at Thr850 is not clear and is evaluated here. Phosphorylation of both Thr695 and Thr850 by Rho-kinase inhibited activity of the type 1 phosphatase catalytic subunit. Rates of phosphorylation of the two sites were similar and efficacy of inhibition following phosphorylation was equivalent for each site. Phosphorylation of each site on MYPT1 was detected in A7r5 cells, but Thr850 was preferred by Rho-kinase and Thr695 was phosphorylated by an unidentified kinase(s).
Impacts
The impact of myosin phosphatase is a critical component of smooth muscle function. Modification of normal phosphatase activity leads to several smooth muscle disorders, including hypertension. Our recent results show that the regulatory mechanism of myosin phosphatase is different from that proposed previously and allows flexibility for regulation by several signal transduction pathways.
Publications
- Muranyi, A., Derkach, D., Erdodi, F., Kiss, A., Ito, M. and Hartshorne, D.J. (2005). Phosphorylation of Thr695 and Thr850 on the myosin phosphatase target subunit: inhibitory effects and occurrence in A7r5 cells. FEBS Lett. 579, 6611-6615.
- Okamoto, R., Kato, T., Mizoguchi, A., Takahashi, N., Nakakuki, T., Mizutani, H., Isaka, N., Imanaka-Yoshida, K., Kaibuchi, K., Lu, Z., Mabuchi, K., Tao, T., Hartshorne, D.J., Nakano, T. and Ito, M. (2005). Characterization and function of MYPT2, a target subunit of myosin phosphatase in heart. Cell. Signal. In Press.
- Lontay, B., Kiss, A., Gergely, P., Hartshorne, D.J. and Erdodi, F. (2005). Okadaic acid influences phosphorylation and translocation of myosin phosphatase target subunit 1 influencing myosin phosphorylation, stress fiber assembly and cell migration in HepG2 cells. Cell. Signal. 17, 1265-1275.
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Progress 01/01/04 to 12/31/04
Outputs
Transient transfection of NIH3T3 cells with various constructs of myosin phosphatase target subunit (MYPT1) and GFP showed distinct cellular localizations. Constructs containing the N-terminal nuclear localization signals (NLS), i.e. full-length MYPT1 and N-terminal MYPT1 fragments, were concentrated in the nucleus. Full-length chicken and human MYPT1-GFP showed discrete nuclear foci. Deletion of the N-terminal NLS or use of central or C-terminal MYPT1 fragments did not show unique nuclear distributions (C-terminal NLS are present). Transient transfection of NIH3T3 cells (in the presence of serum) with full-length MYPT1-GFP caused a marked decrease in number of attached cells, an apparent block in the cell cycle prior to M phase and signs of increased apoptosis. Under conditions of serum starvation the unique nuclear localization of MYPT1-GFP was not found and there was no marked decrease in the number of attached cells (after 48 h). Stable transfection of HEK 293
cells with GFP-MYPT1 was obtained. MYPT1 and its N-terminal mutants bound to retinoblastoma protein (Rb), raising the possibility that Rb is implicated in the effects caused by overexpression of MYPT1.
Impacts
Myosin phosphatase and its target subunit, MYPT1, are involved in dephosphorylation of myosin and thus it was assumed to be co-localized in the cell with myosin. Our recent results show that MYPT1 has several cellular sites, including the nucleus. Accumulation of MYPT1 in the nucleus induces apoptosis and it is therefore suggested that MYPT1, or myosin dephosphorylation, is critical in cell cycle progression from G1 to S phase.
Publications
- Totsukawa, G., Wu, Y., Sasaki, Y., Hartshorne, D. J., Yamakita, Y., Yamashiro, S. and Matsumura, F. (2004). Distinct roles of MLCK and ROCK in the regulation of membrane protrusions and focal adhesion dynamics during cell migration of fibroblasts. J. Cell Biol. 164, 427-439.
- Ito, M., Nakano, T., Erdodi, F. and Hartshorne, D. J. (2004). Myosin phosphatase: structure, regulation and function. Mol.Cell. Biochem. 259, 197-209.
- Wooldridge, A. A., MacDonald, J. A., Erdodi, F., Ma, C., Borman, M. A., Hartshorne, D. J. and Haystead, T. A. J. (2004). Smooth muscle phosphatase is regulated in vivo by exclusion of phosphorylation of threonine 696 of MYPT1 by phosphorylation of Serine 695 in response to cyclic nucleotides. J. Biol. Chem. 279, 34496-34504.
- Lonay, B., Serfozo, Z., Gergely, P., Ito, M., Hartshorne, D. J. and Erdodi, F. (2004). Localization of myosin phosphatase target subunit 1 in rat brain and in primary cultures of neuronal cells. J. Comp. Neurol. 478, 72-87.
- Hartshorne, D. J., Ito, M. and Erdodi, F. (2004). Role of protein phosphatase type 1 in contractile functions. J. Biol. Chem. 279, 37211-37214.
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Progress 01/01/03 to 12/31/03
Outputs
C2C12 cells offer a useful model to study the differentiation of non-muscle cells to skeletal muscle cells.Myosin phosphorylation and changes in related enzymes, with an emphasis on myosin phosphatase were analyzed over the first 6 days of C2C12 differentiation. There was a transition from myosin phosphatase target subunit 1, MYPT1 predominant in the non-muscle cells to increase expression of MYPT2. Levels of MYPT1 or 2 were estimated, and both isoforms were higher in non or partially differentiated cells compared to the concentrations in the differentiated isolated myotubes from day 6. Phosphatase activities, using phosphorylated smooth and skeletal muscle myosins, were estimated for total cell lysates and isolated myotubes. In general, smooth muscle myosin was the preferred substrate.
Impacts
These results suggest that phosphorylation of non-muscle myosin is important in growth of myotubes, either in the fusion process to form larger myotubes or indirectly, by its role in sarcomere organization.
Publications
- Wu, Y., Erdodi, F., Muranyi, A., Nullmeyer, K.D., Lynch, R.M., Hartshorne, D.J. 2003. Myosin phosphatase and myosin phosphorylation in differentiating C2C12 cells. Mus. Res. and Cell Motil. 24:499-511.
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Progress 01/01/02 to 12/31/02
Outputs
A mechanism proposed for regulation of myosin phosphatase (MP) activity is phosphorylation of the myosin phosphatase target subunit (MYPT1). Integrin-linked kinase (ILK) is associated with the contractile machinery and can phosphorylate myosin at the myosin light-chain kinase sites. The possibility that ILK may also phosphorylate and regulate MP was investigated. ILK was associated with the MP holoenzyme, shown by Western blots and in-gel kinase assays. MYPT1 was phosphorylated by ILK and phosphorylation sites in the N- and C-terminal fragments of MYPT1 were detected. From sequence analyses, three sites were identified: a primary site at Thr709 and two other sites at Thr695 and Thr495.
Impacts
The findings that ILK phosphorylated both MYPT1 and myosin and the association of ILK with MP suggest that ILK may influence cytoskeletal structure or function.
Publications
- Muranyi, A., MacDonald, J.A., Deng, J.T., Wilson, D.P., Haystead, T.A.J., Walsh, M.P., Erdodi, F., Kiss, E., Wu, Y. and Hartshorne, D.J. 2002. Phosphorylation of the myosin phosphatase target subunit by integrin-linked kinase. Biochem. J. 366:211-216.
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Progress 01/01/01 to 12/31/01
Outputs
Myotonic dystrophy protein kinase (DMPK) and Rho-kinase are related. An important function of Rho-kinase is to phosphorylate the myosin binding subunit of myosin phosphatase (MYPT1) and inhibit phosphatase activity. Experiments were carried out to determine if DMPK could function similarly. MYPT1 was phosphorylated by DMPK. The phosphorylation site(s) was in the C-terminal part of the molecule. DMPK was not inhibited by the Rho-kinase inhibitors, Y-27632 and HA-1077. Several approaches were taken to determine that a major site of phosphorylation was T654. Phosphorylation at T654 inhibited phosphatase activity. Thus both DMPK and Rho-kinase may regulate myosin II phosphorylation.
Impacts
The activation of Rho kinase and subsequent phosphorylation of the myosin phosphatase target subunit inhibits myosin phosphatase and increases myosin phosphorylation.
Publications
- Muranyi, A., Rongxin, Z., Liu, F., Hirano, K., Ito, M., Epstein, H.F. and Hartshorne, D.J. 2001. Myotonic dystrophy protein kinase phosphorylates the myosin phosphatase targeting subunit and inhibits myosin phosphatase activity. FEBS Letters 493:80-84.
- Yamawaki, K., Ito, M., Machida, H., Moriki, N., Okamoto, R., Isaka, N., Shimpo, H., Kohda, A., Okumura, K., Hartshorne, D.J. and Nakano, T. 2001. Identification of Human CPI-17, an Inhibitory Phosphoprotein for Myosin Phosphatase. Biochem. Biophys. Res. Commun. 285:1040-1045.
- Hartshorne, D.J. 2001. Myosin Light Chain Kinase in "Wiley Encyclopedia of Molecular Medicine. Vol. 3 (ed. by T. Creighton), John Wiley and Sons, Inc. New York, N.Y., pp2197-2200.
- Hartshorne, D.J. 2001. Myosin Phosphatase in "Wiley Encyclopedia of Molecular Medicine. Vol. 3 (ed. by T. Creighton), John Wiley and Sons, Inc. New York, N.Y., pp2200-2203.
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Progress 01/01/00 to 12/31/00
Outputs
Arachidonic acid activates isolated Rho kinase and contracts permeabilized smooth muscle fibres. Various assays were carried out to examine the mechanism of this activation. Native Rho kinase was activated 5 to 6 times by arachidonic acid but an N terminal, constitutively active fragment of Rho kinase, expressed as a glutathione S transferase GST, fusion protein and including the catalytic subunit GST Rho kinase CAT, was not. GST Rho kinase Cat was inhibited by a C terminal fragment of Rho kinase and arachidonic acid removed this inhibition. These results suggest that the C terminal part of Rho kinase, containing the RhoA binding site and the pleckstrin homology domain, acts as an autoinhibitor.
Impacts
The activation of Rho kinase and subsequent phosphorylation of the myosin phosphatase target subunit inhibits myosin phosphatase and increases myosin phosphorylation.
Publications
- MacDonald, J.A., Borman, M.A., Muranyi, A., Somlyo, A.V., Hartshorne, D.J. and Haystead, T.A.J. 2001. Identification of the endogenous smooth muscle myosin phosphatase associated kinase. Proc. Natl. Acad. Sci., U.S.A. 98:2419-2424.
- Butler, T.M., Narayan, S.R., Mooers, S.U., Hartshorne, D.J. and Siegman, M.J. 2001. The myosin cross bridge cycle and its control by twitchin phosphorylation in catch muscle. Biophys. J. 80:415-426.
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Progress 01/01/99 to 12/31/99
Outputs
It is clear from several studies that myosin phosphatase (MP) can be inhibited via a pathway that involves RhoA. However, the mechanism of inhibition is not established. These studies were carried out to test the hypothesis that Rho-kinase (Rho-associated kinase) via phosphorylation of the myosin phosphatase target subunit 1 (MYPT1) inhibited MP activity and to identify relevant sites of phosphorylation. Phosphorylation by Rho-kinase inhibited MP activity and this reflected a decrease in Vmax. Activity of MP with different substrates also was inhibited by phosphorylation. Two major sites of phosphorylation on MYPT1 were Thr695 and Thr850. Various point mutations were designed for these phosphorylation sites. Following thiophosphorylation by Rho-kinase and assays of phosphatase activity it was determined that Thr695 was responsible for inhibition. A site- and phosphorylation-specific antibody was developed for the sequence flanking Thr695 and this recognized only
phosphorylated Thr695 in both native and recombinant MYPT1. Using this antibody it was shown that stimulation of serum- starved Swiss 3T3 cells by lysophosphatidic acid, thought to activate RhoA pathways, induced an increase in Thr695 phosphorylation on MYPT1 and this effect was blocked by a Rho-kinase inhibitor, Y-27632. In summary, these results offer strong support for a physiological role of Rho-kinase in regulation of MP activity.
Impacts
None
Publications
- FENG, J., ITO, M., KUREISHI, Y., ISHIKAWA, K., AMANO, M., ISAKA, N., OKAWA, K., IWAMATSU, A., KAIBUCHI, K., HARTSHORNE, D.J., and NAKANO, T. (1999). Rho-associated kinase of chicken gizzard smooth muscle. J. Biol. Chem. 274, 3744-3752.
- HARTSHORNE, D. J., and HIRANO, K. (1999). Interactions of protein phosphatase type 1, with a focus on myosin phosphatase. Mol. Cell Biochem. 190, 79-84.
- TOTSUKAWA, G., YAMAKITA, Y., YAMASHIRO, S., HOSOYA, H., HARTSHORNE, D. J. and MATSUMURA, F. (1999). Activation of myosin phosphatase targeting subunit by mitosis-specific phosphorylation. J. Cell Biol. 144, 735-744.
- HARTSHORNE, D.J., ICHIKAWA, K., ITO, M., and NAKANO, T. (1999). Structure and regulatory mechanisms of myosin phosphatase, in "Molecular Mechanisms of Smooth Muscle Contraction" (eds. Kohama, K., and Sasaki, Y.) pp. 31-46. E.G. Landes Co., Austin, Texas.
- FENG, J., ITO, M., ICHIKAWA, K., ISAKA, N., NISHIKAWA, M., HARTSHORNE, D.J., and NAKANO, T. (1999). Inhibitory phosphorylation site for Rho-kinase on smooth muscle myosin phosphatase. J. Biol. Chem. 274, 37385-37390.
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Progress 01/01/98 to 12/31/98
Outputs
In the investigation of the sequences of myosin phosphatase target subunit 1 (MYPT1) involved in binding the substrate and catalytic subunit of protein phosphatase type 1 (PP1c), fragments of MYPT1 were prepared and characterized. The shortest fragment capable of full activation of PP1c contained the sequence of residues 1-295. Within this fragment, the N-terminal sequence of residues 1-38 is involved in activation of PP1c (Kcat) and the ankyrin repeats (residues 39-295) were involved in substrate binding (Km). The ankyrin repeats alone (residues 39-295) and the C- terminal fragment of residues 667-1004 did not activate PP1c. Using gel filtration, an interaction with PP1c was detected for the sequences of residues 1-295, 17-295, and 1-170. Affinity columns were prepared with various fragments to assess binding of PP1c. Binding to the column with residues 1-295 was strongest, followed by the binding to the column with residues 1-170. A weak interaction was observed
with the column with residues 1-38. The column with residues 1-295 was used to isolate PP1c from gizzard. The purified PP1c was activated by MYPT1 and fragments to a greater extent than previous preparations. These results suggest that the N- terminal sequence (residues 1-38) and the ankyrin repeats are involved in binding PP1c. The C- terminal ankyrin repeats appear to be dominant, but there is an interaction of PP1c with the N- terminal ankyrin repeats. The N-terminal peptide has two apparent functions, the binding of PP1c via the consensus binding sequence and activation of PP1c by the sequence of residues 1-16.
Impacts
(N/A)
Publications
- Tanaka, J., Ito, M., Feng, J., Ichikawa, K., Hamaguchi, T., Nakamura, M., Hartshorne, D.J., and Nakamo, T. (1998). Interaction of myosin phosphatase target subunit 1 with the catalytic subunit of type 1 protein phosphatase. Biochemistry 37, 16697-16703.
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Progress 01/01/97 to 12/31/97
Outputs
A human brain cDNA library was screened with a cDNA fragment coding for the target subunit of rat myosin phosphatase MYPT1. A cDNA clone representing a novel insoform of MYPT1 was isolated and termed MYPT2. Overlapping clones indicated an open reading frame of 3763 nucleotides and a predicted polypeptide of mass 110,398. Ankyrin repeats and leucine zipper motifs were identified for the sequences 57 316 and 959 982, respectively. Overall, the deduced amino acid sequence of MYPT2 was 61percent identical to MYPT1. Residues 605 660 of MYPT2 were 86 percent identical to the sequence 654 710 of chicken MYPT1 that contains a phosphorylation site thought to be involved in regulation of myosin phosphatase. Western blots, using an antibody specific for MYPT2, showed exclusive expression of MYPT2 in brain and heart. Fragments of MYPT2 representing the N terminal two thirds and C terminal one third were expressed as fusion proteins in E. coli. The N terminal recombinant bound to
the catalytic subunit of type 1 phosphatase isoform and increased activity towards phosphorylated myosin light chain. The C terminal fragment interacted with the 20 kDa subunit of myosin phosphatase. In situ hybridization localized the human MYPT2 gene on chromosome 1q32.1, compared to the chromosomal location 12q 15 q21.2 for MYPT1. These results indicate that MYPT2 is the product of a second gene for the target subunit of myosin phosphatase. It is suggested that the products of the two gene families may be localized differently in various tissues.
Impacts
(N/A)
Publications
- Siegman, M.J., Mooers, S.U., Li, C., Narayan, S., Trinkle Mulcahy, L., Hartshorne, D.J., and Butler, T.M. 1997. Phosphorylation of a high molecular weight 600 kDa protein regulates catch in invertebrate smooth muscle. J. Mus. Res. Cell Motil. 18, 655670.
- Hartshorne, D.J., Ito, M., and Erdodi, F. 1998. Myosin light chain phosphatase. Subunit composition, interactions and regulation. J. Mus. Res. Cell Motil. in press.
- Muranyi, A., Erdodi, F., Ito, M., Gergely, P., and Hartshorne, D.J. 1998. Identification and localization of myosin phosphatase in human platelets. Biochem. J. in press.
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Progress 01/01/96 to 12/30/96
Outputs
Smooth muscle contraction and motile events in none muscle cells is regulated byphosphorylation of myosin. This involves two enzymes, myosin light chain kinase and myosin phosphatase. Myosin phosphatase from smooth muscle consists of a catalytic subunit, PP1c, and two non-catalytic subunits, M130 and M20. Interactions between PP1c, M20 and various mutants of M130 were investigated using the yeast two-hybrid system. PP1c was shown to bind to the N-terminal sequence of M130, 1 to 511. Other interactions were detected, i.e. PP1c to PP1c, M20 to the C-termin M130 were constructed to localize the PP1c and light chain binding regions. Results from the two-hybrid system indicated two binding sites for PP1c on M130. One site in the N-terminal 38 residues and a weaker site(s) in the ankyrin repeats region. Inhibition of PP1c activity with phosphorylase a by the M130 mutants also was consistent with the assignment of these two sites. Overlay assays showed binding of
phosphorylated light chain to the ankyrin repeats, probably in the C-terminal repeats. Activation of PP1c activity with phosphorylated light chain required binding sites for PP1c and substrate, plus an additional sequence C-terminal to the ankyrin repeats. Thus, activation of phosphatase activity, binding of PP1c and substrate are properties of the N-terminal one third of M130.
Impacts
(N/A)
Publications
- HOLDEN, H.M., WESENBERG, G., RAYNES, D.A., HARTSHORNE, D. J., GUERRIERO, V., and RAYMENT, I. (1996). Structure of a proteolytic fragment of TLP20. Acta Crystallogr. 52, 1153-1160.
- ERDODI, F., ITO, M., and HARTSHORNE, D.J. (1996). Myosin Light Chain Phosphate in "Biochemistry of Smooth Muscle Contraction" (Barany, M. Ed.) pp.131-142, Academic Press, San Diego, CA.
- NISHIYAMA, U., UBUKATA, M., MAGAE, J., KATAOKA, T., ERDODI, F., HARTSHORNE, D. J., ISONO, K., NAGAI, K., and OSADA, H. (1996). Structure-activity relationship within a series of degradation products of tautomycin. Biosci. Biotech. Biochem., ICHIKAWA, K., ITO, M., and HARTSHORNE, D.J. (1996). Phosphorylation of the largesubunit of myosin phosphatase and inhibition of phosphatase activity. J. Biol. Chem. 271, 4733-4740.
- ICHIKAWA, K., HIRANO, K., ITO, M., TANAKA, J., NAKANO,T., and HARTSHORNE, D.J. (1996). Interactions and properties of the smooth muscle myosin phosphatase. Biochemistry, 35, 6313-6320.
- HIRANO, K., ERDODI, F., PATTON, J. G., and HARTSHORNE, D.J. (1996). Interaction of protein phosphatas type 1 with a splicing factor. FEBS Lett. 389, 191-194.
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Progress 01/01/95 to 12/30/95
Outputs
The two-hybrid system was used to screen for phosphatase type 1 binding proteins. The cDNA of the delta isoform of the catalytic subunit of type 1 phosphatase was used as bait to screen a chicken gizzard cDNA library. One of the positive clones was similar to a segment of the polypyrimidine tract-binding protein associated splicing factor, PSF. A derived amino acid sequence was determined and corresponded to residues 348 to 589 of human PSF. The amino acid identity was 97.1 percent and the nucleotide identity was 81 percent. This PSF fragment was expressed as a hexahistidine-tagged protein in E. coli and purified. The recombinant PSF fragment inhibited type 1 phosphatase activity both in the presence and absence of Co. Type 1 phosphatase preparations used were: the catalytic subunit from turkey gizzard and from rabbit skeletal muscle; recombinant delta isoform of type 1 catalytic subunit; and the trimeric myosin-bound phosphatase. The catalytic subunit of bovine type
2A phosphatase was not inhibited. Inhibition of type 1 phosphatase activity provided independent evidence for the interaction of the PSF fragment and the catalytic subunit of type 1 phosphatase. In an attempt to localize the binding site, the PSF fragment was cleaved at cysteine 431 to generate an N-terminal peptide, 349 to 430, plus the hexahistidine tag, and a C-terminal peptide, 431 to 589. The N-terminal peptide retained inhibitory activity.
Impacts
(N/A)
Publications
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Progress 01/01/94 to 12/30/94
Outputs
A myosin phosphatase was purified from chicken gizzard smooth muscle. The holoenzyme is a trimer and consists of 130,000-, 38,000-, and 20,000-Da subunits. the catalytic subunit, 38,000 Da, is the type 1# isoform, and its derived amino acid sequence is identical to the rat isoform. The larger subunit bound to myosin and also interacted with the catalytic subunit. cDNA clones encoding the large subunit were isolated from chicken gizzard c DNA libraries. Overlapping clones indicated the presence of two isoforms, and open reading frames of 2889 and 3012 bases were obtained. These encoded proteins of 963 and 1004 amino acids, with masses of 106,700 and 111,600 Da respectively. The insert in the larger isoform is in the center of the molecule, at residues 512-552. The N-terminal third of the molecule is composed of eight repeat sequences, similar to the cdc10/SW16 or ankyrin repeat. Myosin binding and binding to the catalytic subunit are properties of a 58,000-Da fragment
that represents the N-terminal part of the molecule.
Impacts
(N/A)
Publications
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Progress 01/01/93 to 12/30/93
Outputs
Exposure of 3T3-fibroblasts to the phosphatase inhibitor, calyculin-A, induces marked morphological changes and the formation of a aggregate of actin and myosin connected to the nucleus by intermediate filaments. Vimentin was isolated from this complex and shown to be phosphorylated. At least 4 phosphorylation sites were indicated. These sites were distinct from those phosphorylated by the cAMP-dependent protein kinase. Limited proteolysis was used to define the domains in which phosphorylation occurred. Vimentin was isolated from (superscript 32)P-labeled calyculin-A-treated cells and digested with thrombin and (alpha)-chymotrypsin. Proteolysis with thrombin limited the phosphorylation to either the central core or C-terminal domain. Proteolysis with (alpha)-chymotrypsin indicated that the multiple phosphorylation sites were restricted to the C-terminal domain of vimentin.
Impacts
(N/A)
Publications
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Progress 01/01/92 to 12/30/92
Outputs
Calponin is a protein found on the thin filaments of smooth muscle and in the stress fibers of non-muscle cells. It has been proposed as an alternative regulatory mechanism to the well-established myosin phosphorylation system. These studies investigated the mechanism of action of calponin. Calponin inhibits the actin-activated ATPase of smooth muscle myosin and thus has been proposed as a thin filament-based regulatory component in smooth muscle. To obtain information on the mechanism of inhibition by calponin we have used chemical modification of actin and cross-linking of actin and subfragment 1. Modification of Lys 61 of actin had no effect on the inhibition by calponin of acto-heavy meromyosin ATPase, i.e. different from tropomyosin-troponin. In addition, modification of the acidic N-terminal region of actin did not impair the ability of calponin to bind to F-actin. Finally, calponin was effective in inhibiting ATPase activity of cross-linked actosubfragment 1.
Therefore the mechanism of inhibition by calponin is distinct from troponin-tropomyosin and caldesmon in that it does not involve either the N-terminal acidic region of actin nor the area around Lys 61 and does not fit a simple steric blocking model.
Impacts
(N/A)
Publications
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Progress 01/01/91 to 12/30/91
Outputs
Addition of the protein phosphatase inhibitor, calyculin-A, to 3T3 fibroblasts causes a marked change in cell morphology. Initially the cells become rounded, develop surface blebs and then detach from the substratum. In the detached cells an unusual ball-like structure is observed. This study focuses on the cytoskeleton during these CL-A-induced morphological changes. Stress fibers disappear as the cells begin to round and aggregates of actin are formed toward the apical surface of the cell. These aggregates condense, in the detached cells, to form the ball structure of approximately 3 (mu)m diameter. Between the ball and the nucleus are cables of intermediate filaments that appear to be attached to the surface of the ball and to the nuclear lamina. Using a procedure designed for the isolation of nuclei the nucleus-ball complex can be obtained. Analysis of the nucleus-ball preparation by immunofluorescence and electron microscopy demonstrate that the ball contains
actin and that intermediate filaments are located between the ball and the nucleus. In this preparation, the intermediate filaments also appear to attach to the surfaces of the ball and the nucleus. Electrophoretic analysis of the nucleus-ball preparation indicates that, in addition to actin, a major component of the ball is myosin. It is suggested that the formation of the ball is due to an actin-myosin based contractile process, initiated by the phosphorylation of myosin.
Impacts
(N/A)
Publications
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Progress 01/01/90 to 12/30/90
Outputs
Calyculin-A, an inhibitor of type-1 and 2A phosphatases, was applied extracellularly to 3T3 fibroblasts. At 0.1 mu M, calyculin-A caused a market increase in protein phosphorylation in both the cytosolic and insoluble cellular fractions. This effect was independent of external Ca. An immunoprecipitate, formed with an antibody to myosin, contained several cytoskeletal components. Increased phosphorylation following treatment with calyculin-A, was observed in vimentin, the 20-kD myosin light chain and an unidentified 440-kD component. An enhanced level of vimentin phosphorylation was found in intermediate filament preparations from treated cells. Calyculin-A also caused marked shape changes of 3T3 cells. Within minutes after additon of calyculin-A (0.1 mu M) cells became rounded and lost attachment to the substratum. Stress fibers, intermediate filaments and microtubules, prominent in the attached control cells, were not evident in the rounded cells. Shape changes
were reversible and after removal of calyculin-A the rounded cells attached to the substratum, resumed a flattened shape and were active mitotically. In the cells treated with calcyulin-A an unusual "ball-like" structure was observed with transmission electron microscopy. This unique structure was 2 to 3 mu M in diameter and was located close to the nuceus.
Impacts
(N/A)
Publications
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Progress 01/01/89 to 12/30/89
Outputs
Monoclonal antibodies were generated against different regions of the myosin molecule and used to probe function for these regions. Only antibodies raised against S2 (the head-neck junction) influenced biological properties, i.e. the actin-activated Mg ATPase activity, phosphorylation and dephosphorylation rates. Antibodies to S1 (head) and LMM (tail) had no effect. These results indicate that the S2 part of myosin is a determinant of biological properties. The C-terminal domain of MLCK is expressed as an independent protein, termed telokin. It is found only in smooth muscle and its synthesis is hormonally regulated. Peptide homologs of the pseudosubstrate region of MLCK were injected into isolated smooth muscle cells and inhibited contraction. These studies confirm that the major regulatory system in smooth muscle is via the phosphorylation of myosin. Two phosphatase inhibitors have been discovered, okadaic acid and calyculin A. Both promote contraction of
smooth muscle fibers due to inhibition of type 1 and type 2A phosphatases. The two inhibitors differ in their sensitivity to type 1 phosphatase and utilizing this distinction it was shown that the major phosphatase activity in smooth muscle fibers is the type 1 phosphatase. Neither compound activates known kinases and thus it is concluded that even in the relaxed state there is continuous phosphorylation and dephosphorylation of myosin.
Impacts
(N/A)
Publications
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Progress 01/01/88 to 12/30/88
Outputs
Phosphorylation of the two 20,000-dalton light chains of myosin is an important regulatory mechanism in smooth muscle and is required for onset of contraction. The biochemical correllory of contraction is an increase in actin-activated ATPase of myosin. The mechanism through which phosphorylation of the light chains influence active sites on heavy chains is unknown but probably involves a conformational change in head and head-neck regions of the myosin molecule. This "shape activity" hypothesis was challenged by use of monoclonal antibodies generated to different regions of the myosin molecule. Only anti-S2 (neck) antibodies altered biological properties of myosin. properties monitored included: Ca(superscript 2+), Mg(superscript 2+) - and actin-activated ATPase. Rates of phosphorylation and dephosphorylation and limited proteolysis by papain. Antibodies to S1 units (heads) and LMM (tail) did not influence the behavior of myosin. These support the "shapeactivity"
hypothesis and indicate that alterations in the S2 region of myosin are detected by the active site (in S1) and lead to changes in biological properties. Other studies have examined the domain structure of myosin light chain kinase (MLCK). By use of various proteolytic enzymes, differential cleavage of MLCK has been obtained. Cleavage sites were identified by sequence determinations. These provided a detailed domain structure for MLCK and are important in that they define more precisely the pseudosubstrate domain.
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Progress 01/01/87 to 12/30/87
Outputs
The most important of the regulatory mechanisms in smooth muscle is the phosphorylation of myosin, essential for activation of contractile apparatus. Two regulatory enzymes are involved: a myosin light chain kinase and a myosin light chain phosphatase. Kinases have shown that distinct functional domains can be identified, namely catalytic, pseudo-substrate and calmodulin-binding domains. Our aim is to characterize these zones to squence and biochemical properties and then design synthetic peptides that can be used as probes of the in-vivo function of kinase. The other goal is to define the molecular changes that occur on phosphorylation of the light chains. Hypothesis: Critical changes form part of h 10S-6S transition and two areas of the molecule, i.e. the S2 region and a region 68-kDa from the N-terminus of heavy chain, have been identified. How these regions are involved in determining biological properties is our aim and will require a detailed knowledge of the
protein-protein (subunit) interactions. Protein kinase C phosphorylates three residues on the 20,000-dalton light chain, serines 1 and 2 and threonine 9, results in inhibition of ATPase activity and thus the interactions of this light chain region are unique and will complement the data obtained with the myosin light chain kinase. Using limited proteolysis, monoclonal antibodies and kinetic analyses, we intend to probe the structure of the myosin heads to gain more information on the role of myosin phosphorylation in smooth muscle.
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Progress 01/01/86 to 12/30/86
Outputs
Regulation of the contractile apparatus in smooth muscle involves phosphorylation of the myosin light chains. The function of phosphorylation is to convert an inactive to an active species and thereby initiate contraction. Our studies have suggested that the conformation of myosin differs in the active and inactive states and that phosphorylation induces a conformational change. Our present objective is to locate the site(s) of any critical conformational change in the myosin molecule. Previously it was found that an important region is the S1-S2 junction. Using proteolysis by Staph. aureus protease as a conformational probe it is now shown that an additional region of the molecule is affected by the 10S-6S transition. This is tentatively identified as the actin binding site and it is masked in the 10S state and exposed in the 6S state. Consistent with this finding is the observation that actin binding to 10S myosin is of lower affinity than binding to 6S myosin.
These results indicate that several regions of the myosin head are altered during the conformational transition. It is important to identify each of these regions and assign a functional distinction for each conformer. In other studies on the myosin light chain kinase it has been shown that this enzyme can catalyze a reverse reaction and form ATP from phosphorylated light chain and ADP. The formation of ATP adds a new consideration for the energetics of smooth muscle contraction.
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Progress 01/01/85 to 12/30/85
Outputs
Regulation in smooth muscle involves phosphorylation of myosin. The simplest explanation for the role of phosphorylation is that phosphorylation converts an inactive to an active molecule and pertinent activity is actin-activated ATPase. Phosphorylation acts as an "on-off" switch and this implies that the conformation of myosin is different in the phosphorylated and dephosphorylated state. Studies have centered on trying to define the nature of this change. Several approaches have been used, but the most useful is that of limited proteolysis. It was shown (Ikebe and Hartshorne, J. Biol. Chem. 259 11639 (1984)) that the folded form of myosin, referred to as 10S, is more resistent to proteolysis than the extended species, referred to as 6S. Site of the resistent area was shown to be the 1 - 2 junction and this is significant since this part of the molecule influences the mobility of the myosin heads. It was shown that Ca interacts with myosin and induces a
conformational change, that can be detected using limited proteolysis. The Ca effect acts as a modulator on phosphorylated myosin and can increase ATPase activity when Ca-binding sites are saturated. We have found that the myosin light chain kinase, enzyme responsible for phosphorylation of myosin, can insert a second phosphate group into the 20,000-dalton light chain. This effect is additive to single phosphorylation with respect to ATPase activity and the doubly phosphorylated myosin is more firmly locked into the 6S conformation.
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Progress 01/01/84 to 12/30/84
Outputs
Regulation of the contractile apparatus in smooth muscle involves phosphorylation of the 20,000-dalton light chains of the myosin. The critical aspect is that phosphorylation induces a conformation change in myosin, and converts inactive-dephosphorylated myosin to the active-phosphorylated species. Investigations have focussed on defining the nature and extent of this conformation and to establish whether we can demonstrate the effect in intact muscle fibers, rather than in the more convenient in vitro systems. One approach has been to investigate the effects of high MgyRG concentrations using isolated contractile proteins, i.e. myosin and actin. Increasing concentrations of MgC1(2) mimic effects of myosin phosphorylation and induce inactive-active transition of myosin. This proved to be a valuable tool with skinned smooth muscle fitness and tension could be developed at high concentrations of MgC1(2) (i.e. difference 8 mM) and in the absence of myosin
phosphorylation. Another approach has been to study the rates of limited proteolysis of myosin. Inactive myosin species, (IOS), is more resistant to proteolysis with papain than active myosin species, referred to as 6S. When filamentous myosin (which approximates the in-situ condition) is used, two conformations can again be detected, strong evidence that changes observed in-vitro also occur in intact muscle. Our objectives are to continue along these lines.
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Progress 01/01/83 to 12/30/83
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Smooth muscle myosin will, under appropriate solvent conditions, change from theconventional extended form to a folded configuration. This remarkable conformational change does not occur with skeletal and cardicac muscle myosins. We have been studying this effect and have established some of the parameters governing the transition. MgyRG are particularly important in determining myosin shape. However, the most interesting observation is that the conformational change is accompanied by an alteration in the enzymatic properites of myosin, and the hypothesis was developed that the shape of the molecule dictates its biological properties. In addition, it was shown that the ATP-induced tryptophan fluorescence of myosin is also influenced by the myosin conformation, and from the stoichiometry of ATP to myosin, it is possible to conclude that ATP binding to the active sites generates the flourescence signal. From this data, it is possible to infer that the active sites may
be directly modified by the conformation of the myosin molecule. We are continuing to work on this concept, and if valid, it would help to understand the regulatory mechanism in smooth muscle.
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Progress 01/01/82 to 12/30/82
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Considerable progress has been made in elucidating the biochemical mechanisms involved in regulation of the contractile apparatus of smooth muscle. The most popular hypothesis is that the actomyosin system is activated by phosphorylation of myosin involving myosin light-chain kinase, and that deactivation involves removal of the phosphate groups via myosin light chain phosphatase. Although there is considerable experimental evidence to support this hypothesis, there is still some uncertainty about whether the phosphorylation reaction is the only regulatory mechanism or if an additional mechanism is involved. Our recent results suggest that the phosphorylation of myosin is in fact the dominant regulatory pathway. This conclusion was reached by use of a Ca 2 + -independent myosin light chain kinase. Brief proteolysis of the Ca 2 + -dependent form produces an active fragment of M(r) 80,000 that does not require calmodulin. When this fragment is added to skinned
smooth muscle fibers, tension is developed in the absence of Ca 2 + and this is initiated by phosphorylation of myosin. Clearly, since Ca 2 + is not present, other Ca 2 + -requiring mechanisms would not be activated.
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Progress 01/01/81 to 12/30/81
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Our investigation into the mechanism of regulation in smooth muscle has continued. It was confirmed that activation of myosin ATPase activity as a result of light-chain phosphorylation required that both heads of the myosin molecule be phosphorylated. Myosin in which only 1 site is phosphorylated is not markedly activated by actin. The phosphorylation process itself also exhibited a negative cooperativity in that the first head is phosphorylated relatively easily but the second head is much more resistant to phosphorylation. The cooperative phosphorylation is not restricted to smooth muscle, and skeletal muscle myosin exhibits the same pattern of phosphorylation. We wish to extend these studies to include cardiac and non-muscle myosins. In other studies, we have developed a new procedure for the isolation of gizzard myosin light-chain kinase. This method is much more rapid than those used previously, and as a result the extent of proteolytic cleavage of myosin
light-chain kinase apoenzyme is considerably reduced. We feel that the properties of this enzyme will more accurately reflect those occurring in vivo. A Ca 2 +-independent form of the myosin light-chain kinase was also isolated. This preparation is particularly useful because it can be used to assess the significance of myosin phosphorylation in muscle function without complications arising from other Ca 2 +-dependent processes.
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Progress 01/01/80 to 12/30/80
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We have continued to study the Ca 2 +-regulatory mechanism in smooth muscle. Inparticular, we are interested in the relationship of the phosphorylation of myosin to regulation. Over the last year, we have made two significant discoveries. The first of these is that phosphorylation of myosin alone is not adequate to account for the full activation of the smooth muscle actomyosin ATPase activity. Other factors are required and are, therefore, implicated in the regulatory mechanism of smooth muscle. We are currently trying to identify these factors. The second finding is that the partial activation of ATPase activity that is accomplished by phosphorylation is only achieved when both of the myosin heads (active sites) are phosphorylated. Myosin in which only one head is phosphorylated does not exhibit a significant level of actin-activated ATPase activity. This finding is particularly interesting when the physiology of smooth muscle is correlated to the biochemical
results, and it is possible that the myosin with only one phosphorylated site could correspond to the non-cycling cross-bridges, which are thought to be responsible for the unusual physiological behavior of smooth muscle.
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Progress 07/01/79 to 12/30/79
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The research we are engaged in concerns the basic mechanism of smooth muscle contraction and relaxation. Smooth muscle is intimately concerned with the functioning of veins, arteries, gastrointestinal system, uterus, etc. and therefore, is vital to the processes of life. Recently we have isolated and characterized the enzyme which is responsible for the activation by Ca 2 + of the contractile system. The enzyme, myosin light chain kinase, phosphorylates the myosin molecule and this event allows the subsequent activation of the Mg 2 +-ATPase activity by actin. In the muscle this results in the development of tension, or shortening. The rate that phosphorylation can occur and the ATP requirements have been established in-vitro and these will be compared to in-vivo whole muscle data.
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