Progress 10/01/08 to 09/30/13
Outputs
Target Audience: This work will be of general interest to molecular biologists, biochemists, and cell biologists interested in in the role of protein phosphatases in mediating signaling mechanisms. Our studies on the Ser/Thr protein phosphatase PP2a will be generally useful to those researchers interested in the role of calcium in controlling protein phosphorylation via protein phosphatases and in modulating energy homeostasis in cells. Recent work on the Cdc14 phosphatases will be important to those investigating cell cycle regulation by protein phosphorylation and of particular interest to those studying exit from mitosis or the coordination of chromosome segregation with cytokinesis. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? The collaborative project with Dr. Gerrard provided an important research training opportunity for Mr. Park of the Gerrard lab and our collaborative work with Dr. Hall as provided research training opportunities for Christie Eissler and several other students in this lab. How have the results been disseminated to communities of interest? Our research results were reported in a scientific manuscript that has been published in an internationally-recognized, peer-reviewed journal. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
IMPACT: A major goal of our work is to define the physiologic function of Ser/Thr phosphatases by identifying their substrates and defining the mechanisms that regulate them. In a collaborative study with Dr. Gerrard (Animal Sciences, Purdue Univ.) we showed that the Ser/Thr phosphatase PP2A bearing the PR72 regulatory subunit is controlled by changes in calcium permitting it to negatively regulate the AMP-dependent protein kinase (AMPK) of skeletal muscle. AMPK is a crucial regulator of metabolism that signals the cellular need for ATP by responding to increases in the ratio of AMP/ATP. How AMPK responds to the AMP/ATP ratio ( a reflection of the energy state of the cell), upstream protein kinases, and under most circumstances calcium, has been thoroughly studied and is well understood. However, precisely how AMPK is negatively controlled by dephosphorylation mediated by Ser/Thr phosphatases is one aspect of AMPK regulation that remains unclear. Our collaborative work is important because it uncovered a novel mechanism for the negative regulation of AMPK by the calcium-dependent activation of a heterotrimeric form of PP2a containing the PR72 beta subunit, which can bind calcium. The role of calcium in controlling AMPK in muscle is complex but the mechanism for negatively regulating AMPK via PP2a may be important in mediating the adaptation of muscle energy metabolism to conditions where calcium is chronically high. Moreover, there are diseases that induce high calcium levels where the mechanism we have documented may be crucial in controlling muscle metabolism. The Cdc14 phosphatase we have been studying in budding yeast plays a crucial role in coordinating the segregation of chromosomes during mitosis and in properly initiating cytokinesis. Many of the specific functions that Cdc14 plays in yeast and in vertebrates are not yet well defined largely because we do not know the identity of its substrates. The work we have done to precisely define the phosphorylation sites that are specifically recognized by Cdc14 is proving to be important in identifying new candidate substrates. Using criteria based on the substrate specificity defined in our work, we have used a bioinformatics approach to screen the yeast genome and have obtained important lists of candidate substrates. We have also proposed that the unique and restricted selectivity of Cdc14 could provide a basis for precisely controlling the order and timing of events during late mitosis. Better understanding how Cdc14 acts in late mitosis and early cytokinesis will contribute to a more thorough understanding of mechanisms that maintain genome stability. Failure of somatic cells to maintain a stable genome during cell division is a major contributor to tumor formation. Cancer is a genetic disease that results from the accumulation of errors in critical genes. Keeping the genome of a somatic cell intact is required to minimize the chance of developing cancer. When cells undergo cell division, there are several stages of the process where the risks of having their genomes severely altered are high. One crucial stage of cell division is mitosis when the duplicated chromosomes of the cell must be separated and delivered intact to each daughter cell. Failure to accurately execute the segregation of chromosomes at mitosis can result in the gain or loss of either intact or partial chromosomes. Fully defining the role of Cdc14 in maintaining genome stability will contribute to better understanding how cells prevent errant events that damage their chromosomes and drive cancer formation. Outcomes: Objective 1A and 2: Overall goals of this project were to identify novel substrates for the PP5 Ser/Thr phosphatase and to understand how it is regulated. Substrate identification is a key to defining how protein phosphatases function in cell signaling pathways. Recently, through a collaborative effort with Dr. Gerrard (currently Va Tech; formerly Purdue Animal Sciences), we have contributed to identifying a substrate and function of the Ser/Thr phosphatase PP2A in skeletal muscle. The catalytic subunit of PP2A is closely related to PP5 and a member of the same protein phosphatase superfamily. The AMP-dependent protein kinase (AMPK) is a master regulator of energy metabolism in most cells and is regulated allosterically by AMP binding and by phosphorylation of Thr172 in the activation loop of the AMPK alpha subunit. LKB1 and the calcium/calmodulin-dependent protein kinase kinase (CaMKK) are thought to play major roles in phosphorylating and activating AMPK in skeletal muscle. When activated, AMPK enhances glucose uptake and stimulates the β-oxidation of fatty acids. The control of AMPK activity by calcium in skeletal muscle is complex. When calcium concentrations are sustained at high levels over long periods of time (by caffeine or use of calcium ionophores) the activity of AMPK activity decreases substantially. In these collaborative studies, we found that PP2A mediates the inactivation of AMPK in response to long-term elevation of calcium levels. The beta subunit PR72, which is known to possess calcium-binding sites, conferred calcium sensitivity to PP2A and was necessary for the suppression of AMPK activity. This work reveals the crucial role that the PP2A heterotrimer containing PR72 plays in regulating AMPK activity and the metabolism of skeletal muscle. This study has been published in the journal Cell Calcium. Objective 1A: In previous studies for this project performed in collaboration with Dr. Mark Hall, we established new crucial features of the substrate specificity of the Cdc14 phosphatases. We found these features could form the basis for screening coding regions of genomes for candidate phosphorylation sites that would be optimally recognized by Cdc14. Using these sites and a few additional criteria, we identified numerous candidate protein substrates from budding yeast. We believe the validity of this approach has been established. One of the candidate substrates identified in this manner has recently been independently validated as an authentic Cdc14 substrate in a completely separate study recently completed in Dr. Hall’s lab. We have been focused on a set of candidate yeast substrates that are thought be important in initiating or coordinating cytokinesis. Together with Dr. Hall, we have been planning and preparing grants to federal agencies seeking funding for studies to analyze these candidate substrates and the role of Cdc14 in cytokinesis. Thus far, these proposals have not received funding and this has hampered progress in this direction. Similarly, we have been planning, developing, and seeking funding for studies to identify substrates of the two human orthologs of budding yeast Cdc14. A major roadblock to delineating the function of the two human enzymes is the lack of knowledge of its substrates.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
S. Park, T.L. Scheffler, S.S. Rossie, and D.E. Gerrard (2013) AMPK activity is regulated by calcium-mediated protein phosphatase 2A activity. Cell Calcium 53: 217 223
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Progress 10/01/11 to 09/30/12
Outputs
OUTPUTS: A major focus of this project has been to define the substrate selectivity of the Cdc14 phosphatases. This analysis has been completed and was published in the Journal of Biological Chemistry in January 2012. Our manuscript was selected as a "paper of the week" by the editors of the journal indicating that it was considered to be in the top one percent of the more than 6,600 manuscripts published each year in terms of significance and overall importance. In this paper, we report that enzymes of the Cdc14 phosphatase family selectively hydrolyze phosphoserines at sites targeted by the cyclin-dependent kinases (Cdks) in vitro. In the case of budding yeast Cdc14, we provide evidence that the selectivity occurs in cells and is physiologically relevant. We suggest that by discriminating among Cdk sites Cdc14 plays a special role in controlling the order and timing of Cdk site dephosphorylation as cells terminate mitosis. A major goal of this project is to identify novel targets of the two human Cdc14 phosphatases. Our work on the substrate selectivity of Cdc14 is proving to be important in pursuing this goal. Using a bioinformatics approach to identify candidate Cdc14 substrates, we worked together with Dr. Hall to screen all budding yeast proteins for phosphosites matching the specificity of Cdc14. Several of the predicted substrates identified by this screen have known or potential roles in cytokinesis, the process that physically separates daughter and mother cells in late mitosis. Consistent with these results, considerable evidence implicates budding yeast Cdc14 in promoting cytokinesis. However, the role of Cdc14 in cytokinesis is poorly defined in part because only a few of the substrates targeted by Cdc14 during this process are known. In collaboration with Dr. Hall, we have initiated studies to determine if Cdc14 targets these candidate substrates at cytokinesis. The potential substrate Boi1, which is known to be involved in ensuring chromosome segregation is completed before cytokinesis, is of particular interest since it is also a known Cdk substrate and was previously shown to interact with a substrate-trapping mutant of yeast Cdc14 in a two-hybrid screen performed in our lab. The use of the substrate selectivity for identifying new Cdc14 targets will be validated in yeast and then what is learned in these studies will facilitate the application of this approach to the two human Cdc14 orthologs. Importantly, there is evidence that the role of Cdc14 in cytokinesis might be one of the few functions of the budding yeast phosphatase that is conserved in vertebrates. Budding yeast Cdc14 is recruited to the bud neck, the site where cytokinesis takes place. Thus, in related studies, we will define the mechanism by which Cdc14 is recruited to the site of cytokinesis. For these studies, we are creating a series of Cdc14 truncation mutants that are GFP-tagged for localization by fluorescence microscopy. These mutants will be used to identify molecular determinants within Cdc14 required for its bud neck localization. PARTICIPANTS: Collaborators: Drs. Mark C. Hall, Associate Professor, Department of Biochemistry, Purdue University; Laurie Louise Parker, Assistant Professor, Medicinal Chemistry and Molecular Pharmacology, Purdue University; Hana Hall, Associate Research Scientist, Hall Lab, Department of Biochemistry, Purdue University. Graduate student training: Steven Bremmer (has completed PhD), Juan Martinez (has completed PhD), Christie Eissler, and Tom Hinrichsen. TARGET AUDIENCES: This work will be of general interest to molecular biologists, biochemists, and cell biologists interested in cell cycle regulation by protein phosphorylation and of particular interest to those studying exit from mitosis or the coordination of chromosome segregation with cytokinesis. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
For about fifteen years, Cdc14 phosphatases have been thought to act in opposition to cyclin-dependent kinases (Cdks) by dephosphorylating either phosphoserine (pSer) or phosphothreonine (pThr) residues in its substrates. In the collaborative studies completed during this project period, we showed that in vitro yeast Cdc14 possesses a strong preference for pSer over pThr at Cdk sites. We also provided evidence that this substrate specificity is conserved among all Cdc14 phosphatases and revealed how key structural features of the active site account in part for the ability of the enzyme to discriminate between pSer and pThr residues. Studies performed in budding yeast, suggest that the selectivity observed in our in vitro analyses is also exhibited in cells and is of physiologic relevance. These unexpected findings forced a refinement in the current dogma and revealed that Cdc14 phosphatases may act only on a subset of Cdk sites. The intrinsic selectivity of Cdc14 we have uncovered is a novel property of the enzyme that will be important in fully delineating the function of the yeast enzyme and its human orthologs. Moreover, it suggests a potential mechanism explaining how the order of Cdk substrate dephosphorylation could be established during mitotic exit in yeast. The ordered dephosphorylation of Cdk sites in yeast ensures that key events of mitotic exit as well as cytokinesis are properly coordinated. We have recently used the high selectivity in a bioinformatic-screen of the budding yeast genome to predict a set of new candidate substrates of Cdc14 that have been implicated in cytokinesis, a process regulated by Cdc14 but by unknown mechanisms. We intend to help delineate the function of Cdc14 in promoting the onset of cytokinesis by defining the proteins it regulates by dephosphorylation. Errors in chromosome segregation during mitosis or failure to ensure that chromosome segregation is fully completed prior to the initiation of cytokinesis can result in serious errors in the transmission of genetic information to daughter cells and lead to cell death or aneuploid cells. Maintaining genome integrity is important for the prevention of disease and the survival of all organisms. Our work will contribute to elucidating mechanisms that are required for maintaining genome stability during mitosis and cytokinesis and will be of interest to researchers studying the control of cell division.
Publications
- S. C. Bremmer, H. Hall, J. S. Martinez, C. L. Eissler, T. H. Hinrichsen, S. Rossie, L. L. Parker, M. C. Hall, and H. Charbonneau. (2012) Cdc14 Phosphatases Preferentially Dephosphorylate a Subset of Cyclin-dependent kinase (Cdk) Sites Containing Phosphoserine. J. Biol. Chem. 287:1662-1669
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Progress 10/01/10 to 09/30/11
Outputs
OUTPUTS: During the past several years, we have been carefully analyzing the substrate selectivity of the Cdc14 phosphatases in collaboration with Drs. L. Parker and M. Hall. We have discovered that budding yeast Cdc14 exhibits high selectivity for phosphoserine at cyclin-dependent kinase (Cdk) sites. Cdc14 phosphatases were assumed to target both phosphoserine (pSer) or phosphothreonine (pThr) in Cdk sites. Thus our findings indicate the dogma is incorrect and has important implications for cell cycle regulation, particularly exit from mitosis. As reported last year, initial studies suggested this selectivity was shared with the two human Cdc14 orthologs. A major focus over this past year was to complete detailed analyses of the substrate preference of both the human Cdc14A and Cdc14B forms. This work has been finished and unambiguously establishes that both human forms have a strong preference for pSer similar to that observed with the yeast enzyme. Upon completion of the analyses of the two human phosphatases, we prepared a manuscript describing our unexpected findings that was submitted to the Journal of Biological Chemistry. The reviewers requested evidence that the selectivity occurs in vivo. We have now obtained data showing that in yeast cells Cdc14 targets its substrate Acm1 only if it has pSer at its Cdk sites. An Acm1 mutant having pThr at its Cdk sites is refractory to binding to a Cdc14 substrate-trapping mutant and to dephosphorylation in cells. We are now preparing a revised version of this manuscript that will be resubmitted to JBC. To explore residues C-terminal to the pSer influenced substrate selectivity, we synthesized a phosphopeptide library based on the sequence surrounding Pds1pS71, one of the few rigorously mapped in vivo Cdc14 substrate sites. For this analysis, we examined the effects of Lys/Arg residues throughout the +2 to +6 region. We confirmed that peptides containing a Lys at +3 were excellent substrates and found that a single basic residue at only the +3 position supports strong activity. We plan to generate other libraries of peptide variants to identify other sites and residues that influence substrate selection by Cdc14. To examine the ability of Cdc14 to selectively target pSer in recombinant phosphoprotein substrates we needed to monitor the phosphorylation status of individual sites in these proteins. In collaboration with M. Hall and his student C. Eissler, we helped to develop a strategy for examining multiple phosphorylation sites in a single protein using selected reaction monitoring mass spectrometry. A paper describing this method has been published in Analytical Biochemistry. PARTICIPANTS: Collaborators: Mark C. Hall, Department of Biochemsitry, Purdue University; Laurie Louise Parker, MCMP, Purdue University; Graduate student training: Steven Bremmer, Christie Eissler, Juan Martinez TARGET AUDIENCES: Molecular Biologists and Biochemists, particularly those interested in cell cycle regulation by protein phosphorylation or exit from mitosis PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
We have now established that a conserved property of Cdc14 phosphatases is their unexpected ability to discriminate between Cdk phosphorylation sites, by preferentially targeting phosphoserine (pSer) over phosphothreonine (pThr). This property of Cdc14 reveals new complexity in the reversal of Cdk phosphorylation and has important implications for cell cycle control. The mechanisms that control temporal reversal of mitotic phosphorylation events are poorly understood. Our results suggest Cdc14 has specialized roles in reversing Cdk phosphorylation, by working with other phosphatases. We propose the specificity of Cdc14 provides one important mechanism for controlling the timing and fidelity of Cdk site dephosphorylation, ensuring events required for chromosome segregation and cytokinesis are properly coordinated. Importantly, to ensure that their genome is stable and properly transmitted to new generations of cells, each time a cell divides chromosome segregation and cytokinesis must be accurately executed with proper timing. Our findings will be crucial to determining how these cellular events are orchestrated and will be important to other researchers studying mechanisms controlling cell division. In addition, the Cdc14 specificity we report can be used to predict candidate substrates and reveal new functions for human enzymes in cell division and other processes.
Publications
- C. L. Eissler, S. C. Bremmer, J. S. Martinez, L. L. Parker, H. Charbonneau, and M. C. Hall (2011) A general strategy for studying multisite protein phosphorylation using label-free selected reaction monitoring mass spectrometry. Anal Biochem. 418:267-75.
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Progress 10/01/09 to 09/30/10
Outputs
OUTPUTS: In a continued effort to define regulatory protein partners and substrates for PP5 regulation we performed phosphoproteomics analysis of proteins and their phosphorylation status when complexed with active or inactive forms of PP5 heterologously expressed in cells. Elevated levels of heat shock proteins 70 and 90, as well as heat shock protein organizing protein (HOP) were complexed with inactive PP5 as compared to active PP5. We also observed known and novel phosphorylation sites for heath shock protein 90 complexed with inactive PP5. These results suggest that PP5 activity may enhance the transfer of complexes from heat shock protein 70 to heat shock protein 90, and that PP5 may control the phosphorylation status of heat shock protein 90. Heat shock protein 70 is predicted to bind PP5 in a manner similar to heat shock protein 90, through its C terminal "EVVD" TPR binding motif. If so, like heat shock protein 90, heat shock protein 70 should increase PP5 activity. We showed that recombinant heat shock protein 70 increases PP5 activity, comparable to heat shock protein 90. This suggests that PP5 is activated when complexed to either heat shock protein 70 or 90, and may potentially dephosphorylate client or chaperones in early as well as late folding chaperone complexes. In collaboration with Drs. L. Parker and M. Hall, we are continuing our analyses of the substrate selectivity of the Cdc14 phosphatases. In budding yeast, Cdc14 is required to terminate mitosis and to coordinate the onset of cytokinesis. In performing these functions, Cdc14 opposes the cyclin-dependent kinases (Cdks) that trigger the onset of mitosis by phosphorylating Ser-Pro or Thr-Pro sequences. Previously, we showed that Cdc14 exhibits a high degree of selectivity for pSer-Pro sites (p designates a phosphorylated residue), but has very low or no activity toward pThr-Pro sequences suggesting that Cdc14 acts on a subset of sites targeted by Cdks. In the past year, we have thoroughly examined the substrate selectivity of the human Cdc14A and Cdc14B enzymes and have confirmed that their selectivity is comparable to that of the yeast enzyme establishing that the preference for pSer-Pro sites is conserved among all Cdc14 phosphatases. We have also examined the importance of basic residues at sites C-terminal to the pSer-Pro residues. A single basic residue (Lys or Arg) at the +3 position (pSer-Pro-xxx-Lys/Arg) greatly enhances the ability of these substrates to be dephosphorylated by Cdc14. Adding basic residues at +4 and +5 further improved the ability of substrates to be dephosphorylated. The structure of human Cdc14B reveals the basis for the preferential targeting of substrates with basic residues C-terminal to the Pro residue. HCdc14B has a groove containing negatively charged side chains located near the active site in a position where they could accommodate and interact with the C-terminal basic residues of a protein substrate. These studies are revealing a previously unrecognized selectivity of Cdc14 for a subset of Cdk sites and will be important in fully understanding how Cdks regulate cell division. PARTICIPANTS: Collaborators: David L. Armstrong, National Institute of Enviromental Health Sciences, Research Triangle Park, NC; Feng Yang and Richard D. Smith, Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA; Mark C. Hall, Department of Biochemsitry, Purdue University; Laurie Louise Parker, MCMP, Purdue University; Graduate student training: Steven Bremmer, Christie Eissler, Ayesha Elias TARGET AUDIENCES: Biochemists PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Our studies suggest that PP5 may influence protein maturation by controlling phosphorylation of client proteins undergoing folding in chaperone complexes containing heat shock protein 70 and 90 by dephosphorylating clients or chaperone complex components. Heat shock proteins mediate fundamental cell stress responses and control the phosphorylation and activation status of many signaling kinases and transcription factors, and are key targets for therapeutic intervention in cancer and other disease processes. Defining the function of PP5 in heat shock protein 70 and 90 chaperone function may reveal how these proteins are regulated in normal and stressed or diseased cells and may provide insights into moderating their activity in disease. We have uncovered a previously unrecognized preference of Cdc14 for pSer-Pro sequences having basic residues at the +3 position and have shown that this selectivity is shared by all Cdc14 phosphatases including the human Cdc14 enzymes. This finding shows that Cdk sites can be differentially regulated during the cell cycle. It is clear that the differences in the time at which Cdk phosphorylation sites are removed after anaphase provides a means to ensure chromosome segregation, exit from mitosis, and cytokinesis occur with the proper order and timing. Thus, our work reveals that Cdc14 has the ability to play a major role in determining the order in which sites targeted by Cdks are removed as cells complete mitosis. This study is currently being prepared for publication. The publication of this work will have a major impact by enhancing our understanding of mechanisms by which Cdk-dependent phosphorylation controls progression of cells through mitosis.
Publications
- No publications reported this period
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Progress 10/01/08 to 09/30/09
Outputs
OUTPUTS: In one study on the regulation of PP5 activity, we used immunoprecipitation, in vitro binding, cellular fractionation, and immunofluorescence techniques to show that protein phosphatase 5 (PP5) interacts specifically and directly with an active form of the monomeric G protein Rac1. Activation of Rac1 promoted PP5 translocation to the plasma membrane in intact cells and stimulated PP5 phosphatase activity in vitro. PP5 has been implicated in regulating cellular responses to DNA damage, but thus far only one substrate, DNA-dependent protein kinase, has been identified. To define targets for PP5 regulation, in a second study we performed a phosphoproteomics analysis of proteins whose phosphorylation status changed as a function of PP5 activity during the DNA damage response. Six targets for PP5 regulation were identified. Of these, four are known to participate in DNA damage responses. Bioinformatics analysis suggested that PP5 regulatory targets are important in regulation of translation and in the dephosphorylation of substrates for kinases involved in cell cycle control and cell proliferation. In a third study we demonstrated that PP5 can protect neurons from the toxic effects of amyloid beta peptide or treatment with reactive oxygen species, and that neurons with reduced levels of PP5 are more vulnerable to these toxic treatments. In a separate study, we have examined the substrate preference of the Cdc14 phosphatases that coordinate chromosome segregation, mitotic spindle dynamics, termination of mitosis, and cytokinesis. In executing these functions, Cdc14 is thought to oppose cyclin-dependent kinases (Cdks) by dephosphorylating the proline-directed sites (Thr/Ser-Pro) targeted by these enzymes. These models have never been confirmed by direct biochemical analyses of substrates. In collaboration with Drs. Laurie Parker and Mark Hall, we have synthesized phosphopeptide substrates based on known Cdc14 targets to examine its substrate selectivity in vitro. We have discovered that Cdc14 selectively targets Ser with a catalytic efficiency at least 1000-fold higher than that for Thr. Substitution of the Pro at the +1 position with Ala results in a 500-fold reduction in catalytic efficiency confirming the selectivity for Pro-directed sites targeted by Cdks. We have found that in vitro Cdc14 selectively targets Ser in physiologically relevant full length proteins and have preliminary results indicating this selectivity is observed in vivo. Importantly, Cdc14 orthologs from fission yeast and humans exhibit nearly identical ability to differentiate between Ser and Thr suggesting the substrate selectively of this phosphatase is evolutionarily conserved. An active site mutant of Cdc14, in which Ala 285 is replaced by Gly, increases the size of the active site and partially relaxes the selectivity for Ser suggesting that one mechanism by which the enzyme discriminates between Ser and Thr involves differences in the size of their side chains. PARTICIPANTS: Collaborators: David L. Armstrong, National Institute of Environmental Health Sciences, Research Triangle Park, NC; Feng Yang and Richard D. Smith, Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA; Mark C Hall, Department of Biochemistry, Purdue University; Laurie Louise Parker, MCMP, Purdue University; Graduate Student Training: Efrain Sanchez-Ortiz, Hemalatha Jayachandran, Anindya Chatterjee, Steven Bremmer, Christie Eissler TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Little is known about the function, regulation or targets for PP5 in cellular responses. We previously established that Rac1 requires PP5 for stimulation of Kv11.1 channels. Our new study has defined how Rac1 regulates PP5. In the case of the DNA damage response, we identified six previously unknown targets for PP5 regulation, however it remains to be determined if these are direct substrates for PP5 or are indirectly regulated by PP5 action. This knowledge helps further define the role of PP5 in the DNA damage response, which is a key protective pathway for preventing the inappropriate proliferation of cells containing excessively damaged DNA, as can occur in cancer. Our third study implicates PP5 as a neuroprotective enzyme in diseases arising from oxidative stress. Accumulation of cellular damage by oxidative stress is considered a major underlying cause of many diseases of aging, as well as acute conditions such as stroke and heart attacks. Thus our studies have advanced our understanding of PP5 regulation, function and targets of PP5 action. Our studies of substrate preference indicate that Cdc14 is highly selective and targets a restricted subset of sites phosphorylated by cyclin-dependent kinases (Cdks). This work has revealed previously unrecognized complexity in the regulation of Cdk targets and highlights a mechanism, by which specific Cdk sites can be differentially regulated. These findings have important implications for understanding the role of Cdks in cell cycle control.
Publications
- Sanchez-Ortiz, E, Hahm, BK, Armstrong DL, Rossie, S. (2009). Protein phosphatase 5 protects neurons against amyloid-beta toxicity. J Neurochem. 111:391-402.
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