Progress 10/01/03 to 09/30/08
Outputs
OUTPUTS: Our goal is to understand the function and regulation of protein phosphates 5 (PP5) in hormonal signal transduction and in cellular stress responses. Previously we found that in several regions containing major populations of glucocorticoid receptor-expressing neurons, PP5 and glucocorticoid receptors are co-localized at the cellular level. We have now shown that PP5 and glucocorticoid receptors are associated in complexes isolated from brain tissue. We are investigating the regulation of glucocorticoid receptor by PP5 in neurons using central nervous system-derived cell lines and in cultured primary neurons. We are also investigating the role of PP5 in protecting neurons from oxidative stress. We are also applying phosphoproteomic approaches to identify PP5 substrates. PARTICIPANTS: Hemalatha Jayachandran Ayesha Elias Ling Wang Efrain Sanchez-Ortiz Anindya Chaterjee TARGET AUDIENCES: Biochemists and researchers studying alzheimers disease. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
In collaboration with Armstrong and colleagues, we previously found that thyroid hormone stimulated, Rac-mediated activation of KCNH2 potassium channels in GH4C1 pituitary cells requires PP5 (Gentile et al, PNAS 2006: 103, 5202). We have now shown that Rac specifically and directly binds PP5 in vitro, and in cells recruits PP5 to the plasma membrane. This suggests that one mechanism by which Rac promotes PP5 regulation of KCNH2 channels is by translocating PP5 to the plasma membrane where it can act on target ion channels (manuscript in preparation). We also found that overexpression of active, but not inactive, PP5 prevents neuronal death induced by H2O2 and by amyloid beta, the major toxic component of amyloid plaques in brains of Alzheimer's patients. This suggests that PP5 may play a protective role against neuronal cell death induced by amyloid regulator in Alzheimer's disease (manuscript in preparation). We performed 3 proteomics analyses to identify additional potential PP5 targets and regulators. In one study, heat shock proteins 70 and 90, as well numerous co-chaperones, were complexed with PP5, suggesting that PP5 plays a role in chaperone function from the early stages of protein folding through late stages of protein maturation. We also identified a novel phosphorylation site on heat shock protein 90, a potential target of PP5 action. Protein kinase A, centaurin and DARPP32 are complexed with PP5 in brain extracts, suggesting potential neuronal roles for PP5 in regulation of cyclic AMP responses, dopaminergic neuron function and neuronal vesicle trafficking. PP5 has been implicated in the DNA damage response, but targets of dephosphorylation are largely known. In HeLa cells treated with bleomycin to induce DNA damage, we found that phosphorylation levels of four DNA damage response proteins changed as a function of PP5 activity. These proteins represent potential PP5 targets during activation of the DNA damage pathway (manuscript submitted).
Publications
- Ham, B.M., Yang, F., Jayachandran, H., Jaitly, N., Monroe, M.E., Gritsenko, M.A., Livesay, E.A., Zhao, R., Purvine, S.O., Orton, D., Adkins, J.N., Camp 2nd, D.G., Rossie, S. and Smith, R.D. 2008. The influence of sample preparation and replicate analyses on HeLa Cell phosphoproteome coverage. J. Proteome Res. 7:2215-2221.
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Progress 10/01/06 to 09/30/07
Outputs
OUTPUTS: Our goal is to understand the function and regulation of protein phosphatase 5 (PP5) in hormonal signal transduction and in cellular stress responses. Previously we found that in several regions containing major populations of glucocorticoid receptor-expressing neurons, PP5 and glucocorticoid receptors are co-localized at the cellular level. We have now shown that PP5 and glucocorticoid receptors are associated in complexes isolated from brain. We are investigating the regulation ofglucocorticoid receptor by PP5 in neurons using central nervous system-derived cell lines and in cultured primary neurons. We are also investigating the role of PP5 in protecting neurons from oxidative stress. We are also applying phosphoproteomic approaches to identify PP5 substrates.
PARTICIPANTS: Hemalatha Jayachandran: currently a predoctoral student. She works full time in my lab and is suported by my NIH grant. She prepared samples for proteomics analysis, and validated numerous proteomic targets that were identified. This work was done in collaboration with the Pacific Northwest National Laboratory.
TARGET AUDIENCES: Biochemists
Impacts
We have developed a new phosphoproteomic strategy to identify substrates for protein phosphatase regulation. This new method can be used to analyze tissues that cannot be easily quantified by current phosphoproteomic strategies. Our method is applicable to very small samples and tissues that cannot be labeled in culture, such as human biopsied samples.
Publications
- No publications reported this period
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Progress 10/01/05 to 09/30/06
Outputs
Our goal is to understand the function and regulation of protein phosphatase 5 (PP5) in hormonal signal transduction and in cellular stress responses. Previously we found that PP5 and glucocorticoid receptors were co-localized in distinct regions of rat brain. We went on to determine whether these two proteins are co-localized at the cellular level. While glucocorticoid receptors are present in both neurons and glial cells throughout the brain, PP5 is primarily expressed in neurons. In several regions containing major populations of glucocorticoid receptor-expressing neurons, PP5 and glucocorticoid receptors are co-localized at the cellular level. There are also neuronal populations containing glucocorticoid receptors where PP5 is either absent or below detection limits. Likewise, numerous neuronal populations contain PP5, but not glucocorticoid receptors. These studies suggest that glucocorticoid
receptors may be differentially regulated by phosphatase action in different populations of central neurons. Co-localization of PP5 and glucocorticoid receptors occurred in all brain regions involved in feedback control of the hypothalamus-pituitary-adrenal axis. Thus, PP5 may be an important modulator of glucocorticoid receptor responses in this pathway. We have initiated experiments to determine if PP5 controls glucocorticoid receptor function in neurons using central nervous system-derived cell lines and in cultured primary neurons. In collaboration with Armstrong and colleagues, we found that PP5 modulates a specific type of potassium channel via a unique hormone signaling pathway. We are now investigating the specific role of PP5 in this pathway, since it has the potential to reveal both regulators and substrates for PP5, and to define a role for PP5 in hormone and signaling pathways that are
associated with a number of human disease states. We also examined the role of PP5 in activation of the tumor suppressor p53 by okadaic acid, an environmental toxin, tumor promoter and specific inhibitor of S/T phosphatases including PP5. In contrast to results in cancer cell lines, we found that in non-neoplastic cells, blockade of PP5 did not activate p53. Instead, okadaic acid activated p53 via blockade of a related enzyme, PP2A. Finally, we are involved in proteomics studies in collaboration with Richard Smith and colleagues to define cellular substrates for PP5. This requires developing new strategies to overcome challenging problems in proteomics analysis. Two studies were performed to optimize conditions for global cellular phosphoproteome analysis, one to increase sensitivity and quantitation and to reduce the sample size required for analysis, and one to optimize methods to quantify changes in
phosphorylation during phosphatase inhibition. Building on these studies, we are now applying phosphoproteomics to define the role of PP5 in DNA damage repair pathways and in heat shock protein 90 chaperone function.
Impacts
We have shown that PP5 mediates hormonal regulation of ion channel function. The signaling pathway in which PP5 participates involves thyroid hormone, a G protein implicated in neuronal development and in cancer cell metastasis, and a potassium channel implicated in genetically based cardiac arrhythmias; thus defining the relation of PP5 to each of these pathway elements can have important implications for human disease states. PP5 has been also implicated in controlling glucocorticoid receptor function in non-neuronal cells. Our localization studies also implicate PP5 in modulating neuronal glucocorticoid receptors, and consequently, central nervous system responses to stress. We also showed that PP5 is not the primary target for the cellular toxicity associated with the environmental toxin and tumor promoter okadaic acid. Our ongoing investigations are directed at defining the molecular details
concerning the role of PP5 in hormonal regulation of a potassium channel, identification of PP5 substrates and regulators, and the potential role of PP5 in disease states associated with environmental stress, and with development and aging processes. Our proteomics studies will provide technical advances that should be of general use to analyze changes in cellular protein phosphorylation, which is essential for a complete understanding of nearly all signal transduction pathways.
Publications
- Messner DJ, Romeo C, Boynton A and Rossie S. 2006. Inhibition of PP2A, but not PP5, mediates p53 activation by low levels of okadaic acid in rat liver epithelial cells. J. Cellular Biochem. 99:241-55.
- Rossie S, Jayachandran H and Meisel RL. 2006. Cellular Co-localization of Protein Phosphatase 5 and Glucocorticoid Receptors in Rat Brain. Brain Research 1111:1-11.
- Luo Q, Tang K, Yang F, Elias A, Shen Y, Moore RJ, Zhao R, Hixson KK, Rossie, SS, Smith RD. 2006. More sensitive and quantitative proteomic measurements using very low flow rate porous silica monolithic LC columns with electrospray ionization-mass spectrometry. J. Proteome Res. 5:1091-7
- Gentile S, Darden T, Romeo C, Russo A, Martijn N, Erxleben C, Rossie S, Armstrong DL. 2006. Rac GTPase Signaling through the PP5 Ser/Thr phosphatase. Proc. Nat. Acad. Sci. 103: 5202-5206.
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Progress 10/01/04 to 09/30/05
Outputs
Our long-term goals are to understand the function and regulation of protein phosphatase 5 (PP5) in the central nervous system. Previously we reported that PP5 and glucocorticoid receptors (GR) were co-localized in some, but not all cell populations in distinct regions of rat brain. These studies were extended to show that PP5 is expressed primarily in neurons of adult rat brain, since it was co-localized with h-Nu, a neuronal marker, but not with glial acidic fibrillar protein, a glial cell marker. As expected, GR was found in both cell populations. These findings are now being prepared for publication. We are now initiating experiments to determine if PP5 controls GR function in neurons using central nervous system-derived cell lines. We previously reported that PP5 dephosphorylates tau, which is found in a hyperphosphorylated state in neurofibrillary tangles characteristic of diseased neurons in Alzheimer's patients. To further investigate the relevance of tau
dephosphorylation by PP5 to Alzheimer's disease, we demonstrated that PP5 acted on specific tau sites known to be hyperphosphorylated in Alzheimer's disease, showed that the specific activity of PP5 toward phospho-tau in vitro was comparable to that of an established tau phosphatase, PP2A, and showed that PP5 activity was decreased in the neocortex of postmortem brains of Alzheimer's patients. This recently published study supports a role for PP5 as an important tau phosphatase, the activity of which may be compromised in Alzheimer's disease. We failed to find a link between PP5 and regulation of BK potassium channels, one of our original specific aims. Instead, however, in collaboration with Armstrong and colleagues, we obtained evidence of a role for PP5 in modulation of another type of potassium channel via a unique hormone signaling pathway (Gentile et al, manuscript submitted). We are now investigating the specific role of PP5 in this pathway, since it has the potential to reveal
an activator and a substrate for PP5, and to define a role for PP5 in hormone and signaling pathways that are associated with a number of human disease states. We also investigated the role of PP5 in activation of the tumor suppressor p53 by okadaic acid, which is an environmental toxin, tumor promoter and specific inhibitor of ser/thr phosphatases including PP5. In contrast to results reported for cancer cell lines, we found that in non-neoplastic cells, blockade of PP5 did not activate p53. Instead, we found okadaic acid activated p53 via blockade of a related enzyme PP2A (manuscript in preparation). Finally, we are involved in ongoing proteomics studies in collaboration with Richard Smith and colleagues to define cellular substrates for PP5. These studies require developing new strategies to overcome challenging problems in proteomics analysis. Some of our progress in efforts to increase sensitivity and reduce sample size required for analysis is included in a manuscript now being
prepared for publication. We also collaborated with Dr. Scott McLuckey's group in a recently published study exploring new mass spectrometry methods for identifying whole proteins in complex mixtures.
Impacts
Our work has shown that PP5 is involved in mediating hormonal regulation of ion channel function and implicates PP5 as an important phosphatase for tau, which is hyperphosphorylated and concentrated in neurofibrillary tangles in diseased neurons of Alzheimer's patients. We also showed that PP5 is unlikely to be the primary target for the cellular toxicity associated with the environmental toxin and tumor promoter okadaic acid. Our ongoing investigations are directed toward defining the molecular details concerning the role of PP5 in hormonal regulation of a potassium channel, identification of PP5 substrates, and the potential role of PP5 in disease states associated with environmental stress, and with development and aging processes. Our ongoing proteomics studies will provide technical advances that should be of general use to analyze changes in cellular protein phosphorylation, which is essential for a complete understanding of nearly all signal transduction
pathways.
Publications
- Yan F, He M, Hogan JM, Rossie SS and McLuckey SA. 2005. Targeted Biomarker Detection via Whole Protein Ion Trap Tandem Mass Spectrometry: Thymosin 4 in a Human Lung Cancer Cell Line. J. Mass. Spectrom. 40: 444-451.
- Liu F, Iqbal K, Grundke-Iqbal I, Rossie S and Gong C-X. 2005. Dephosphorylation of Tau by Protein Phosphatase 5: Impairment in Alzheimer Disease. J. Biol. Chem. 280: 1790-1796.
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Progress 10/01/03 to 09/29/04
Outputs
We are using molecular biology and biochemistry to determine the role of PP5 in hormone and neurotransmitter signal transduction. We purified a lipid-stimulated protein Ser/Thr phosphatase, PP5, from bovine brain. PP5 contains a catalytic domain similar to those of PP1 and PP2A, and an N-terminal TPR (Tetratricopeptide Repeat) domain which typically mediates protein-protein interactions. We found that the TPR domain acts with the C-terminal region to inhibit PP5 and the binding of unsaturated fatty acids to the TPR domain or C-terminal truncation of 10-13 residues relieves inhibition in vitro. This suggests that PP5 is an auto-inhibited enzyme and that the binding of a protein and/or lipid to the TPR domain may regulate PP5 activity in vivo. The TPR domain, by binding specific proteins, may also control substrate specificity and subcellular localization of PP5. To reveal potential functions for PP5 in the central nervous system, we identified specific brain regions
that express high levels of PP5, including hippocampus, cortex, cerebellum, striatum, and the hypothalamus. This suggests PP5 may be involved in neuroendocrine secretion, memory formation or motor function, and also indicated that PP5 shares overlapping regional distribution with type II glucocorticoid receptors in brain. Since these receptors are putative targets for PP5 regulation in non-neuronal cells and are critical in nervous system development, stress responses and aging, we are now investigating whether PP5 and glucocorticoid receptors are co-localized at the cellular level within these brain regions. In collaborative studies with Dr. Cheng-Xin Gong (New York State Institute for Basic Research in Developmental Disabilities) we have shown that PP5 may be an important tau phosphatase in brain. Since tau phosphorylation is thought to be important in the neurodegenerative processes underlying Alzheimers disease, these studies suggest that PP5 activity in neurons could help prevent
the degenerative processes that lead to this debilitating disease. We have also initiated a proteomics study to identify cellular targets for PP5 in neurons and endocrine cells. Using a yeast 2 hybrid screen, we identified 3 novel potential binding partners for PP5; 2 of these proteins have features found in transcriptional regulators, and the third contains multiple repeated protein binding motifs found in scaffolding proteins. We are now determining whether these proteins bind PP5 in cells, and the functional relationship between these proteins and PP5. We have developed a mutant form of PP5 which is relatively insensitive to phosphatase inhibitors. We are currently using this mutant to examine the role of PP5 in regulation of the tumor suppressor p53 and other signal transduction pathways that control cell growth, differentiation or programmed cell death (apoptosis). We have also made cell lines expressing this mutant for the purpose of identifying novel substrates for PP5 in
neuronal and cancer cell lines.
Impacts
The information learned in these studies increases our understanding of how cellular excitation is controlled. The regulatory enzymes we have identified may be new drug targets for controlling ion channel function in the treatment of nervous, cardiovascular or endocrine disorders. In addition, these enzymes are also attacked by certain natural environmental toxins. Our work showing that these enzymes play an essential role in ion channel regulation reveals a potential path by which these environmental toxins may compromise nerve, muscle or endocrine cell function.
Publications
- Gong, C-X, Liu, F., Wu, G., Rossie, S., Wegiel, J., Li, L., Grundke-Iqbal, I., and Igbal, K. 2004 Dephosphorylation of microtubule-associated protein tau by protein phosphatase 5 J of Neurochem 88: 298-310.
- Manaves V, Wuxuan Q, Bauer AL, Rossie S, Kobayashi M, and Rane S. 2004 Calcium and Vitamin D increase mRNA levels for the growth control hIKI channel in human epiderman keratinocytes but functional channels are not observed BMC Dermatology 4:7
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Progress 10/01/02 to 09/30/03
Outputs
We are using molecular biology and biochemistry to determine the role of PP5 in hormone and neurotransmitter signal transduction.We purified a lipid-stimulated protein Ser/Thr phosphatase, PP5, from bovine brain. PP5 contains a catalytic domain similar to those of PP1 and PP2A, and an N-terminal TPR(Tetratricopeptide Repeat)domain which typically mediates protein-protein interactions.We found that the TPR domain acts with the C-terminal region to inhibit PP5 and the binding of unsaturated fatty acids to the TPR domain or C-terminal truncation of 10-13 residues relieves inhibition in vitro.This suggests that PP5 is an auto-inhibited enzyme and that the binding of a protein and/or lipid to the TPR domain may regulate PP5 activity in vivo.The TPR domain, by binding specific proteins, may also control substrate specificity and subcellular localization of PP5.In order to understand the role of PP5 in brain, we mapped the regional distribution of this enzyme.PP5
immunofluorescence was observed in the cerebral cortex,cerebellum,and the supraoptic nucleus of the hypothalamus,globus pallidus,hippocampus,thalamus,lateral preoptic area of the hypothalamus,and substantia nigra.Staining occurred primarily in perikarya and proximal processesThis differs from other reports showing PP5 is primarily nuclear in cultured, dividing cells, and suggests the subcellular localization of PP5 may change during cell growth or differentiation.The widespread distribution of PP5 suggests it serves diverse functions in brain.We are investigating the potential role of PP5 in 2 pathways important for brain function;stress hormone-mediated changes in neuronal structure and function and regulation of calcium-dependent potassium channels by hormones and neurotransmitters.Using immunofluorescence we have demonstrated that PP5 and glucocorticoid receptors are co-localized at the cellular level in several regions of the brain,including the
hippocampus,cortex,cerebellum,substantia nigra and ponsThis suggests PP5 may regulate glucocorticoid signaling in these brain areas,where this stress hormone impacts higher cognitive functions,emotion and memory,and motor control.These proteins appear to be co-localized in large neuronal cells, whereas glucocorticoid receptors alone are also present in smaller cells that may be glia.To examine this more closely,we are performing co-immunofluorescence studies to determine whether PP5 or glucocorticoid receptors are present in cells expressing glial acidic fibrillar protein or h-Nu,expressed only in neurons.We have initiated a proteomics study to identify cellular targets for PP5 in neurons and endocrine cells.Using a yeast 2 hybrid screen, we identified 3 novel potential binding partners for PP5;2of these proteins have features found in transcriptional regulators, and the third contains multiple repeated protein binding motifs found in scaffolding proteins.We are now determining
whether these proteins bind PP5 in cells,and the functional relationship between these proteins and PP5.Preliminary studies indicate that PP5 can be co-immunoprecipitated with 1 of these proteins,BRL(Bromodomain-like protein)when expressed as an epitope-tagged protein.
Impacts
The information learned in these studies increases our understanding of how cellular excitation is controlled. The regulatory enzymes we have identified may be new drug targets for controlling ion channel function in the treatment of nervous, cardiovascular or endocrine disorders. In addition, these enzymes are also attacked by certain natural environmental toxins. Our work showing that these enzymes play an essential role in ion channel regulation reveals a potential path by which these environmental toxins may compromise nerve, muscle or endocrine cell function.
Publications
- Jeong JY, Johns J. Sinclair C, Park JM, and Rossie S. (2003) Characterization of Saccharomyces cerevisiae protein Ser/Thr phosphatase T1 and comparison to its mammalian homolog PP5. BMC Cell Biol.4:3
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Progress 10/01/01 to 09/30/02
Outputs
Define the role of Ser/Thr protein phosphatase 5 (PP5) in calcium-dependent potassium channel regulation in pituitary-derived GH4C1 cells. 2) Determine if PP5 controls glucocorticoid receptor (GR) function in neurons. 3) Identify substrates for PP5 in neuronal and endocrine cells. APPROACH: We are using molecular biology and biochemistry to determine the role of PP5 in hormone and neurotransmitter signal transduction. PROGRESS: We purified a lipid-stimulated protein Ser/Thr phosphatase, PP5, from bovine brain. PP5 contains a catalytic domain similar to those of PP1 and PP2A, and an N-terminal TPR (Tetratricopeptide Repeat) domain which typically mediates protein-protein interactions. We found that the TPR domain acts together with the C-terminal region to inhibit PP5 and the binding of unsaturated fatty acids to the TPR domain or C-terminal truncation of 10-13 residues relieves inhibition in vitro. This suggests that PP5 is an auto-inhibited enzyme and that the
binding of a protein and/or lipid to the TPR domain may regulate PP5 activity in vivo. The TPR domain, by binding specific proteins, may also control substrate specificity and subcellular localization of PP5. In order to understand the role of PP5 in brain, we mapped the regional distribution of this enzyme. PP5 immunofluorescence was observed in the cerebral cortex, cerebellum, and the supraoptic nucleus of the hypothalamus, globus pallidus, hippocampus, thalamus, lateral preoptic area of the hypothalamus, and substantia nigra. Staining occurred primarily in perikarya and proximal processes. This differs from reports by others showing that PP5 is primarily nuclear in cultured, dividing cells, and suggests the subcellular localization of PP5 may change during cell growth or differentiation. The widespread distribution of PP5 suggests that it serves diverse functions in brain. We are now investigating the potential role of PP5 in two pathways important for brain function; stress
hormone-mediated changes in neuronal structure and function and the regulation of calcium-dependent potassium channels by hormones and neurotransmitters. Preliminary studies show that PP5 and glucocorticoid receptors are co-localized at the cellular level in several regions of the brain, including the hippocampus, cortex, cerebellum and pons. This suggests PP5 may regulate glucocorticoid signaling in these brain areas, where this stress hormone impacts higher cognitive functions, emotion and memory, and motor control. We also have initiated a proteomics study to identify cellular targets for PP5 in neurons and endocrine cells. Using a yeast two hybrid screen, we identified three novel potential binding partners for PP5; two of these proteins have features commonly found in transcriptional regulators, and the third contains multiple repeated protein binding motifs found in scaffolding proteins. We are currently determining whether any of these proteins bind PP5 in cells, and what the
functional relationship between these proteins and PP5 may be.
Impacts
The information learned in these studies increases our understanding of how cellular excitation is controlled. The regulatory enzymes we have identified may be new drug targets for controlling ion channel function in the treatment of nervous, cardiovascular or endocrine disorders. In addition, these enzymes are also attacked by certain natural environmental toxins. Our work showing that these enzymes play an essential role in ion channel regulation reveals a potential path by which these environmental toxins may compromise nerve, muscle or endocrine cell function.
Publications
- Erxleben C, Everhart AL, Romeo C, Florance H, Bauer MB, Alcorta DA, Rossie S, Shipston MJ, Armstrong DL.(2002) Interacting effects of N-terminal variation and strex exon splicing on slo potassium channel regulation by calcium, phosphorylation, and oxidation. J Biol Chem.277:27045-52
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Progress 10/01/00 to 09/30/01
Outputs
OBJECTIVES: 1) Define the role of Ser/Thr protein phosphatase 5 (PP5) in calcium-dependent potassium channel regulation by protein phosphorylation in pituitary-derived GH4C1 cells. 2) Determine if PP5 controls glucocorticoid receptor (GR) function in neurons. 3) Identify novel substrates for PP5 in neuronal and endocrine cells. APPROACH: We are using molecular biology and biochemistry to determine the role of PP5 in hormone and neurotransmitter signal transduction. PROGRESS: We purified a protein Ser/Thr phosphatase, PP5, from bovine brain based on its arachidonic acid-stimulated activity. This enzyme contains a catalytic domain similar to those of PP1, PP2A and calcineurin, and an N-terminal TPR (Tetratricopeptide Repeat) domain which typically mediates protein-protein interactions. We found that the TPR domain acts together with the C-terminal region to inhibit PP5 and the binding of unsaturated fatty acids to the TPR domain or C-terminal truncation of 10-13
residues relieves inhibition in vitro. This suggests that PP5 is an auto-inhibited enzyme and that the binding of a protein and/or lipid to the TPR domain of PP5 may regulate its activity in vivo. Through its protein binding capacity, the TPR domain may also control substrate specificity and subcellular localization in a manner similar to that of the regulatory subunits of related phosphatases. In order to understand the role of PP5 in brain, we mapped the regional distribution of this enzyme. The most intense staining was observed in neurons of the cerebral cortex, cerebellum, and the supraoptic nucleus of the hypothalamus. Other areas containing immunoreactive cells included the globus pallidus, hippocampus, thalamus, lateral preoptic area of the hypothalamus, and substantia nigra. Staining in these cells was observed primarily in perikarya and proximal processes. This differs from reports by others showing that PP5 is primarily nuclear in cultured, dividing cells, and suggests that
the subcellular localization of PP5 may be a function of the state of cell growth or differentiation. The widespread distribution of PP5 suggests that it serves diverse functions in brain. We are now investigating the potential role of PP5 in two pathways important for brain function; stress hormone-mediated changes in neuronal structure and function and the regulation of calcium-dependent potassium channels by hormones and neurotransmitters. We also have initiated a proteomics study to identify cellular targets for PP5 in neurons and endocrine cells. Using a yeast two hybrid screen, we identified three novel potential binding partners for PP5; two of these proteins have features commonly found in transcriptional regulators, and the third contains multiple repeated protein binding motifs found in scaffolding proteins. Scaffolding proteins typically bind several proteins that function in a common signaling or metabolic pathway. We are currently determining whether any of these proteins
bind PP5 in cells, and what the functional relationship between these proteins and PP5 may be
Impacts
The information learned in these studies increases our understanding of how cellular excitation is controlled. The regulatory enzymes we have identified may be new drug targets for controlling ion channel function in the treatment of nervous, cardiovascular or endocrine disorders. In addition, these enzymes are also attacked by certain natural environmental toxins. Our work showing that these enzymes play an essential role in ion channel regulation reveals a potential path by which these environmental toxins may compromise nerve, muscle or endocrine cell function.
Publications
- Bahl, R., Bradley, K. C., Thompson, K. J., Swain, R. A., Rossie, S., and Meisel, R. L. (2001) Localization of Protein Ser/Thr Phosphatase 5 in Rat Brain. Mol Brain Research 90, 101-109.
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Progress 10/01/99 to 09/30/00
Outputs
We purified a protein phosphatase 5 (PP5) on the basis of lipid-activated phosphate activity. This enzyme contains a C-terminal catalytic domain and an N-terminal domain consisting of 3 tetratricopeptide repeats (TPRs). The TPR domain of PP5 mediates its interaction with other proteins, including several hormone receptors. Using limited proteolysis and site-directed mutagenesis, we have found that both the TPR domain and the C terminus control the activation of PP5 by lipid, and that the TPR domain interacts directly with lipids. This suggests that, in addition to binding other proteins, the TPR domain of PP5 may regulate enzyme activity in an autoinhibitory manner. Proteins that bind the TPR domain of PP5 could also potentially activate the enzyme, alone or in combination with lipids. In order to provide a detailed picture of how the TPR domain and C terminus interact with and regulate the catalytic domain of PP5, we have initiated a collaboration with Dr. Lydia
Tabernero (U. Manchester) to determine the crystal structure of this enzyme. None of the proteins currently postulated to bind PP5 are known to activate it. We have employed a yeast two-hybrid screen to identify protein partners that may regulate PP5 or be substrates for this enzyme. This screen has identified two putative interacting proteins, which we are now studying. In collaboration with Dr. Armstrong and his colleagues at NIEHS we are using a molecular biology approach to define the role of PP5 in ion channel regulation. Overexpression of the regulatory domain of PP5 in anterior pituitary-derived GH4Cl cells disrupts their electrophysiological response to the hormone somatostatin. When a mutant form of PP5 with altered sensitivity to specific phosphatase inhibitors such as okadaic acid is expressed in GH4Cl cells, their electrophysiological response to hormonal stimulation is correspondingly altered in its sensitivity to okadaic acid. These findings support a role for PP5 in
mediating ion channel regulation by the hormone somatostatin. PP5 mRNA is widely expressed, but is most abundant in brain. To learn more about the function of PP5 in brain we mapped its distribution in this organ. Our results show that PP5 is expressed in a number of discrete regions, suggesting it may serve diverse functions in brain.
Impacts
(N/A)
Publications
- No publications reported this period
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Progress 10/01/98 to 09/30/99
Outputs
We purified protein phosphatase 5 (PP5) on the basis of lipid-activated phosphatase activity. This enzyme contains a C-terminal catalytic domain and an N-terminal domain consisting of 3 tetratricopeptide repeats (TPRs). TPRs are thought to mediate protein-protein interaction, and several reports suggest that the TPR domain of PP5 mediates its interaction with other proteins,including several hormone receptors. Using limited proteolysis and site-directed mutagenesis, we have found that both the TPR domain and the C.terminus control the activation of PP5 by lipid, and that the TPR domain interacts directly with lipid. This suggests that, in addition to binding other proteins, the TPR domain of PP5 may regulate enzyme activity in an autoinhibitory manner. Proteins that bind PP5's TRP domain could also potentially activate the enzyme, alone or in combination with lipid. In order to provide a detailed picture of how the TPR domain and C. terminus interact with and regulate
the catalytic domain of PP5, we have initiated a collaboration with Dr. Lydia Tabernero (U. Manchester) to determine the crystal structure of this enzyme. None of the proteins currently postulated to bind PP5 are known to activate it. We have employed a yeast two-hybrid screen to identify protein partners that may regulate PP5. This screen has identified one putative interacting protein, which we are now studying. In collaboration with Dr. Armstrong and his colleagues at NIEHS we are using a molecular biology approach to define the role of PP5 in ion channel regulation. Overexpression of the regulatory domain of PP5 in anterior pituitary-derived GH4Cl cells disrupts their electrophysiological response to the hormone somatostatin. When a mutant form of PP5 with altered sensitivity to specific phosphatase inhibitors is expressed in GH4Cl cells, their electrophysiological response to hormonal stimulation is correspondingly altered in its sensitivity to okadaic acid. These findings
support a role for PP5 in mediating ion channel regulation by the hormone somatostatin. PP5 mRNA is widely expressed, but is most abundant in brain. To learn more about the function of PP5 in brain, we have begun immunocytochemical studies to map its distribution. Our results show that PP5 is abundant in cerebellar Purkinje cells of rat brain, suggesting a potential role for this enzyme in the control of cerebellar function.
Impacts
(N/A)
Publications
- No publications reported this period
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Progress 10/01/97 to 09/30/98
Outputs
We purified protein phosphatase 5 (PP5) on the basis of lipid-activated phosphatase activity. This enzyme contains a C-terminal catalytic domain and an N-terminal domain consisting of 3-4 tetratricopeptide repeats (TPRs). TPRs are thought to mediate protein-protein interaction, and several reports suggest that the TPR domain of PP5 mediates its interaction with other proteins, including several hormone receptors. Using limited proteolysis and site-directed mutagenesis, we have found that both the TPR domain and the C-terminus control the activation of PP5 by lipid, and that the TPR domain interacts directly with lipid. This suggest that, in addition to binding other proteins, the TPR domain of PP5 may regulate enzyme activity in an autoinhibitory manner. Proteins that bind PP5's TRP domain could also potentially activate the enzyme, alone or in combination with lipid. None of the proteins shown to interact with PP5 so far appears to alter activity. We have initiated a
project employing yeast two-hybrid analysis to identify interacting proteins that may regulate the activity of PP5. There is currently no structure available for any TPR domain or TPR-containing protein. In order to provide a detailed picture of how the TPR domain and C-terminus interact with and regulate the catalytic domain of PP5, we have initiated a collaboration with Drs. Cynthia Stauffacher and Lydia Tabernero (Biol. Sciences, Purdue U.) to determine the crystal structure of this enzyme. We are using a molecular biology approach to selectively manipulate the expression, activity, or inhibitor sensitivity of PP5. Overexpression of the regulatory domain of PP5 in anterior pituitary-derived GH4Cl cells disrupts their electrophysiological response to the hormone somatostatin. When a mutant form of PP5 with altered sensitivity to specific phosphatase inhibitors is expressed in GH4Cl cells, their electrophysiological response to hormonal stimulation is correspondingly altered in its
sensitivity to okadaic acid. These findings support a role for PP5 in mediating ion channel regulation by the hormone somatostatin. These studies are being performed in collaboration with Dr. Armstrong and his colleagues at NIEHS.
Impacts
(N/A)
Publications
- No publications reported this period
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Progress 10/01/96 to 09/30/97
Outputs
We purified protein phosphatase 5 (PP5) on the basis of lipid-activated phosphatase activity. This enzyme contains a C-terminal catalytic domain and an N-terminal domain consisting of 3-4 tetratricopeptide repeats (TPRs). TPRs are thought to mediate protein-protein interaction, and several reports suggest that the TPR domain of PP5 mediates its interaction with other proteins, including several hormone receptors. Using limited proteolysis and site-directed mutagenesis, we have found that both the TPR domain and the C-terminus control the activation of PP5 by lipid, and that the TPR domain interacts directly with lipid. This suggest that, in addition to binding other proteins, the TPR domain of PP5 may regulate enzyme activity in an autoinhibitory manner. Proteins that bind PP5's TPR domain could also potentially activate the enzyme, alone or in combination with lipid. None of the proteins shown to interact with PP5 so far appears to alter activity. We have initiated a
project employing yeast two-hybrid analysis to identify interacting proteins that may regulate the activity of PP5. There is currently no structure available for any TPR domain or TPR-containing protein. In order to provide a detailed picture of how the TPR domain and C-terminus interact with and regulate the catalytic domain of PP5, we have initiated a collaboration with Drs. Cynthia Stauffacher and Lydia Tabernero (Biol. Sciences, Purdue U.) to determine the crystal structure of this enzyme. We are using a molecular biology approach to selectively manipulate the expression, activity, or inhibitor sensitivity of PP5. Overexpression of the regulatory domain of PP5 in anterior pituitary-derived GH4C1 cells disrupts their electrophysiological response to the hormone somatostatin. When a mutant form of PP5 with altered sensitivity to specific phosphatase inhibitors is expressed in GH4C1 cells, their electrophysiological response to hormonal stimulation is correspondingly altered in its
sensitivity to okadaic acid. These findings support a role for PP5 in mediating ion channel regulation by the hormone somatostatin. These studies are being performed in collaboration with Dr. Armstrong and his colleagues at NIEHS.
Impacts
(N/A)
Publications
- Kondratyuk, T. and Rossie, S. 1997 Depolarization of rat brain synaptosomes increases phosphorylation of voltage-sensitive sodium channels. J. Biol. Chem. 272, 16978-16983.
- Skinner, J., Sinclair, C., Romeo, C., Armstrong, D., Charbonneau, H. and Rossie, S. 1997 Purification of a fatty acid-stimulated protein Ser/Thr phosphatase from bovine brain and its identification as a homolog of protein phosphatase 5. J. Biol. Chem. 272, 22464-22471.
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Progress 10/01/95 to 09/30/96
Outputs
I use a biochemical approach to examine the structure and regulation of ion channels by second messenger systems. One major project is focused on how reversible phosphorylation of voltage-sensitive sodium channels is controlled in brain. The sodium channel is phosphorylated at multiple sites by cyclic AMP-dependent protein kinase and by protein kinase C. Phosphorylation alters sodium channel responses during depolarization and therefore modulates neuronal excitation. We have found that three ser/thr phosphatases dephosphorylate sodium channels in vitro and in situ, calcineurin and two distinct forms of PP2A. Our work with synaptosomes demonstrates that depolarization causes a calcium-dependent PKC-mediated phosphorylation of sodium channels at cAMP-dependent phosphorylation sites. Studies with purified sodium channels suggest that phosphorylation of channels by PKC can decrease their susceptibility to dephosphorylation of cAMP-dependent phosphorylation sites by either
calcineurin or by PP2A. Thus, one mechanism by which depolarization or PKC activation can lead to an increase in phosphorylation at PKA sites may be through a decrease in sodium channel dephosphorylation. This may be an important mechanism for convergent regulation of ion channels and other neuronal phosphoproteins by different signal transduction pathways. We also found that different forms of PP2A from rat brain exhibit different activity toward sodium channels phosphorylated at cAMP-dependent phosphorylation sites.
Impacts
(N/A)
Publications
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Progress 10/01/94 to 09/30/95
Outputs
I use a biochemical approach to examine the structure and regulation of ion channels by second messenger systems. One major project is focused on how reversible phosphorylation of voltage-sensitive sodium channels is controlled in brain. The sodium channel is phosphorylated at multiple sites by cyclic AMP-dependent protein kinase and by protein kinase C. Phosphorylation alters sodium channel responses during depolarization and therefore modulates neuronal excitation. We have shown that phosphatase 2A and the calcium and calmodulin-dependent phosphatase calcineurin dephosphorylate cyclic AMP-dependent phosphorylation sites on sodium channels. We have also shown that during depolarization of synaptosomes, there is an increase in phosphorylation at cyclic AMP-dependent sites. This is not due to an increase in cyclic AMP-dependent kinase activity, but requires calcium influx and protein kinase C. When purified sodium channels are phosphorylated by protein kinase C or by
both protein kinase C and cyclic AMP-dependent protein kinase, they are resistant to dephosphorylation by either phosphatase 2A or by calcineurin. We hypothesize that phosphorylation of sodium channels by protein kinase C somehow renders them resistant to dephosphorylation by phosphatase 2A and calcineurin. We are currently trying to identify the phosphatase(s) responsible for dephosphorylating protein kinase C phosphorylation sites on sodium channels and learn how this phosphatase is regulated. We have also been studying the regula.
Impacts
(N/A)
Publications
- NO PUBLICATIONS REPORTED THIS PERIOD.
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Progress 10/01/93 to 09/30/94
Outputs
My work is focused on structure and regulation of ion channels by second messenger systems. We are studying biochemical aspects of reversible phosphorylation of voltage-sensitive sodium channels in brain. This channel is phosphorylated by cAMP-dependent protein kinase at multiple sites in situ. We have identified and partially purified two protein phosphatase(s) from brain that dephosphorylate sodium channels, phosphatase 2A and calcineurin. Phosphatase 2A appears to exist in multiple forms in brain; interestingly, brain forms of this enzyme are much more active in dephosphorylating sodium channels than is the purified catalytic subunit of phosphatase 2A alone. We have shown that okadaic acid, a potent inhibitor of phosphatase 2A and cyclosporin A, which inhibits calcineurin, enhance sodium channel phosphorylation in synaptosomes. Depolarization of synaptosomes leads to an increase in sodium channel phosphorylation in cAMP-dependent phosphorylation sites. This effect
requires calcium influx and is mediated by protein kinase C. Since phosphorylation decreases sodium channel excitability, this may provide a mechanism for reducing excitation of neurons after a period of stimulation. This would be an important negative feedback mechanism to protect neurons against hyperexcitation.
Impacts
(N/A)
Publications
- No publications reported this period.
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Progress 10/01/92 to 09/30/93
Outputs
My work is focused on structure and regulation of ion channels by second messenger systems. We are studying biochemical aspects of reversible phosphorylation of voltage-sensitive sodium channels in brain. This channel is phosphorylated by cAMP-dependent protein kinase at multiple sites in situ. We have identified and partially purified two protein phosphatase(s) from brain that dephosphorylate sodium channels, phosphatase 2A and calcineurin. Phosphatase 2A appears to exist in multiple forms in brain; interestingly, brain forms of this enzyme are much more active in dephosphorylating sodium channels than is the purified catalytic subunit of phosphatase 2A alone. This implies that some aspect of the brain forms of phosphatase 2A, such as subunit composition or post-translational modification, modulates the activity of the catalytic subunit. We have shown that okadaic acid, a potent inhibitor of phosphatase 2A, enhances sodium channel phosphorylation and the activity of
phosphatase 2A or calcineurin in synaptosomes. We are also investigating whether phosphatase or kinases are closely associated with sodium channels at the membrane. In another project, we are investigating the mechanism by which the hormones somatostatin and atrial natriuretic factor inhibit secretion from anterior pituitary cells. These hormones activate potassium channels in pituitary cells by stimulating a serine/threonine phosphatase.
Impacts
(N/A)
Publications
- No publications reported this period.
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Progress 10/01/91 to 09/30/92
Outputs
My work is focused on structure and regulation of ion channels by second messenger systems. We are studying biochemical aspects of reversible phosphorylation of voltage-sensitive sodium channels in brain. This channel is phosphorylated by cAMP-dependent protein kinase at multiple sites in situ. We have identified and partially purified two protein phosphatase(s) from brain that dephosphorylate sodium channels, phosphatase 2A and calcineurin. Phosphatase 2A appears to exist in multiple forms in brain; interestingly, brain forms of this enzyme are much more active in dephosphorylating sodium channels than is the purified catalytic subunit of phosphatase 2A alone. This implies that some aspect of the brain forms of phosphatase 2A, such as subunit composition or post-translational modification, modulates the activity of the catalytic subunit. We have shown that okadaic acid, a potent inhibitor of phosphatase 2A, enhances sodium channel phosphorylation and the activity of
phosphatase 2A or calcineurin in synaptosomes. We are also investigating whether phosphatase or kinases are closely associated with sodium channels at the membrane. We are also investigating the mechanism by which the hormones somatostatin and atrial natriuretic factor inhibit secretion from anterior pituitary cells. These hormones activate potassium channels in pituitary cells by stimulating a serine/threonine phosphatase.
Impacts
(N/A)
Publications
- NO PUBLICATIONS REPORTED THIS PERIOD.
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