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Patricia T.w. Cohen - One of the best experts on this subject based on the ideXlab platform.
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Erratum to “Ppm1E is an in cellulo AMP-activated Protein kinase Phosphatase” [Cell. Signalling 23 (2011) 114–124]
Cellular Signalling, 2011Co-Authors: Martin Voss, James Paterson, Ian R. Kelsall, Cristina Martin-granados, C. James Hastie, Mark Peggie, Patricia T.w. CohenAbstract:Activation of 5′-AMP-activated Protein kinase (AMPK) is believed to be the mechanism by which the pharmaceuticals, metformin and phenformin, exert their beneficial effects for treatment of type 2 diabetes. These biguanide drugs elevate 5′-AMP, which allosterically activates AMPK and promotes phosphorylation on Thr172 of AMPK catalytic α subunits. Although kinases phosphorylating this site have been identified, Phosphatases that dephosphorylate it are unknown. The aim of this study is to identify Protein Phosphatase(s) that dephosphorylate AMPKα-Thr172within cells. Our initial data indicated that members of the Protein Phosphatase Mg/Mn-dependent DOI of original article: 10.1016/j.cellsig.2010.08.010. ⁎ Corresponding author. Tel.: +44 1382 384240; fax: +44 1382 223778. E-mail address: p.t.w.cohen@dundee.ac.uk (P.T.W. Cohen). 1 These authors have contributed the major experimental data in the article. 0898-6568/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.cellsig.2010.12.001 (PPM) family andnot thoseof thePPP family of Protein serine/threonine Phosphatases may be directly or indirectly inhibited by phenformin. Using antibodies raised to individual Ppm Phosphatases that facilitated the assessment of their activities, phenformin stimulation of cells was found to decrease theMg/Mn-dependent Protein serine/threonine Phosphatase activity of Ppm1E and Ppm1F, but not that attributable to other PPM family members, including Ppm1A/PP2Cα. Depletion of Ppm1E, but not Ppm1A, using lentiviral-mediated stable gene silencing, increased AMPKα-Thr172 phosphorylation approximately three fold in HEK293 cells. In addition, incubation of cells with low concentrations of phenformin and depletion of Ppm1E increased AMPK phosphorylation synergistically. Ppm1E and the closely related Ppm1F interact weakly with AMPK and assays with lysates of cells stably depleted of Ppm1F suggest that this Phosphatase contributes to dephosphorylation of AMPK. The data indicate that Ppm1E and probably PpM1F are in cellulo AMPK Phosphatases and that Ppm1E is a potential anti-diabetic drug target.
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Sorting the Protein Phosphatases: okadaic acid led the way
Biochemical Journal, 2010Co-Authors: Patricia T.w. CohenAbstract:A seminal paper by Corinna Bialojan and Akira Takai [(1988) Biochem. J. 256, 283–290] examined the effects of a compound extracted from marine black sponges, okadaic acid, on Protein Phosphatases. The identification of okadaic acid as a potent inhibitor of particular PPP family Phosphatases was an extremely important milestone, not only in the differentiation of different Protein Phosphatases, but also in processes by which these Protein Phosphatases were identified as in vivo regulators of cellular signalling. In the present article, I review the information that led to the recognition of okadaic acid as a Protein serine/threonine Phosphatase inhibitor, and examine its currently known specificity and uses. I discuss its mode of inhibition and compare its interaction at the catalytic centre with those of more recently identified Protein Phosphatase inhibitors and inhibitory domains. Finally I consider the ongoing process of finding more specific Protein Phosphatase inhibitors.
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Ppm1E is an in cellulo AMP-activated Protein kinase Phosphatase.
Cellular signalling, 2010Co-Authors: Martin Voss, James Paterson, Ian R. Kelsall, Cristina Martin-granados, C. James Hastie, Mark Peggie, Patricia T.w. CohenAbstract:Activation of 5'-AMP-activated Protein kinase (AMPK) is believed to be the mechanism by which the pharmaceuticals, metformin and phenformin, exert their beneficial effects for treatment of type 2 diabetes. These biguanide drugs elevate 5'-AMP, which allosterically activates AMPK and promotes phosphorylation on Thr172 of AMPK catalytic α subunits. Although kinases phosphorylating this site have been identified, Phosphatases that dephosphorylate it are unknown. The aim of this study is to identify Protein Phosphatase(s) that dephosphorylate AMPKα-Thr172 within cells. Our initial data indicated that members of the Protein Phosphatase Mg/Mn(2+)-dependent [corrected] (PPM) family and not those of the PPP family of Protein serine/threonine Phosphatases may be directly or indirectly inhibited by phenformin. Using antibodies raised to individual Ppm Phosphatases that facilitated the assessment of their activities, phenformin stimulation of cells was found to decrease the Mg(2+)/Mn(2+)-dependent [corrected] Protein serine/threonine Phosphatase activity of Ppm1E and Ppm1F, but not that attributable to other PPM family members, including Ppm1A/PP2Cα. Depletion of Ppm1E, but not Ppm1A, using lentiviral-mediated stable gene silencing, increased AMPKα-Thr172 phosphorylation approximately three fold in HEK293 cells. In addition, incubation of cells with low concentrations of phenformin and depletion of Ppm1E increased AMPK phosphorylation synergistically. Ppm1E and the closely related Ppm1F interact weakly with AMPK and assays with lysates of cells stably depleted of Ppm1F suggest [corrected] that this Phosphatase contributes to dephosphorylation of AMPK. The data indicate that Ppm1E and probably PpM1F are in cellulo AMPK Phosphatases and that Ppm1E is a potential anti-diabetic drug target.
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Handbook of Cell Signaling - CHAPTER 100 – Protein Serine/Threonine Phosphatases and the PPP Family
Handbook of Cell Signaling, 2003Co-Authors: Patricia T.w. CohenAbstract:The current classification of Protein serine/threonine Phosphatases is based upon the primary structures of their catalytic subunits. The amino acid sequences of the catalytic subunits fall into two main groups that have been termed the PPP family, of which the prototypic member is Ppplc (PPl), and the PPM family, of which the prototypic member is Ppm1c (PP2C). The Protein serine/threonine Phosphatase hat dephosphorylates the carboxy-terminal domain (CTD) of RNA polymerase II possesses a distinct amino acid sequence and thus represents the founding member of a third family, FCP. Protein serine/threonine Phosphatases are enzymes that reverse the actions of Protein kinases by cleaving phosphate from serine and threonine residues in Proteins. They are structurally and functionally distinct from the acid and alkaline Phosphatases and are also separate from the family of Protein Phosphatases encompassing the tyrosine and dual specificity (tyrosine and serine/threonine) Phosphatases.
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Cloning of a novel testis specific Protein serine/threonine Phosphatase, PPN 58A, from Drosophila melanogaster
Biochimica et Biophysica Acta, 1998Co-Authors: Christopher G. Armstrong, Viktor Dombrádi, David J. Mann, Patricia T.w. CohenAbstract:Abstract A gene encoding a novel member of the PPP family of Protein serine/threonine Phosphatases, termed PPN 58A, was cloned from Drosophila melanogaster . The deduced amino acid sequence of PPN 58A exhibits 59–62% identity to D. melanogaster PP1 isoforms, 51% identity to D. melanogaster PPY 55A and ≤40% identity to other members of the PPP family. The single copy gene PPN 58A maps to chromosome 2 locus 58A . Analysis of PPN 58A mRNA reveals that, like PPY 55A, PPN 58A is a testis specific enzyme.
Brian E Wadzinski - One of the best experts on this subject based on the ideXlab platform.
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Alpha4 is a ubiquitin-binding Protein that regulates Protein serine/threonine Phosphatase 2A ubiquitination.
Biochemistry, 2010Co-Authors: Jamie L. Mcconnell, Guy R. Watkins, Sarah E. Soss, Heidi S. Franz, Lisa R. Mccorvey, Benjamin W. Spiller, Walter J. Chazin, Brian E WadzinskiAbstract:Multiple regulatory mechanisms control the activity of the Protein serine/threonine Phosphatase 2A catalytic subunit (PP2Ac), including post-translational modifications and its association with regulatory subunits and interacting Proteins. Alpha4 is a PP2Ac-interacting Protein that is hypothesized to play a role in PP2Ac ubiquitination via its interaction with the E3 ubiquitin ligase Mid1. In this report, we show that alpha4 serves as a necessary adaptor Protein that provides a binding platform for both PP2Ac and Mid1. We also identify a novel ubiquitin-interacting motif (UIM) within alpha4 (amino acid residues 46−60) and analyze the interaction between alpha4 and ubiquitin using NMR. Consistent with other UIM-containing Proteins, alpha4 is monoubiquitinated. Interestingly, deletion of the UIM within alpha4 enhances its association with polyubiquitinated Proteins. Lastly, we demonstrate that addition of wild-type alpha4 but not an alpha4 UIM deletion mutant suppresses PP2Ac polyubiquitination. Thus, the p...
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Isolation and characterization of PP2A holoenzymes containing FLAG-tagged B subunits.
Methods in molecular biology (Clifton N.J.), 2007Co-Authors: Deanna G. Adams, Brian E WadzinskiAbstract:Protein serine/threonine Phosphatase 2A (PP2A) is a major cellular enzyme implicated in the control of a wide variety of biological processes. The predominant form of PP2A in cells is a heterotrimeric holoenzyme (ABC) consisting of a scaffolding (A) subunit, a regulatory (B) subunit, and a catalytic (C) subunit. Although numerous signal transduction pathways are known to be regulated by PP2A, the identity of the PP2A holoenzymes controlling each pathway remains unclear. Studies aimed at elucidating substrates for individual PP2A holoenzymes have been hindered by the limited availability of purified endogenous holoenzymes and the inability to differentiate cellular roles of closely related PP2A holoenzymes. In this chapter, we describe a strategy for the functional expression of select FLAG-tagged regulatory B subunits in human embryonic kidney-293 cells and subsequent purification of PP2A holoenzymes containing the FLAG-tagged B subunit and endogenous A and C subunits (ABFLAGC). Biochemical analyses of the purified ABFLAGC holoenzymes reveal that they exhibit virtually indistinguishable specific activities and sensitivities to inhibitors as compared to the corresponding endogenous PP2A holoenzymes. The strategy described herein provides a straightforward method to purify individual PP2A holoenzymes from target mammalian cells for subsequent in vitro studies, as well as a powerful approach to identify cellular substrates and roles for each holoenzyme.
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Positive regulation of Raf1-MEK1/2-ERK1/2 signaling by Protein serine/threonine Phosphatase 2A holoenzymes.
The Journal of biological chemistry, 2005Co-Authors: Deanna G. Adams, R. Lane Coffee, Hong Zhang, Steven L. Pelech, Stefan Strack, Brian E WadzinskiAbstract:Abstract Protein serine/threonine Phosphatase 2A (PP2A) regulates a wide variety of cellular signal transduction pathways. The predominant form of PP2A in cells is a heterotrimeric holoenzyme consisting of a scaffolding (A) subunit, a regulatory (B) subunit, and a catalytic (C) subunit. Although PP2A is known to regulate Raf1-MEK1/2-ERK1/2 signaling at multiple steps in this pathway, the specific PP2A holoenzymes involved remain unclear. To address this question, we established tetracycline-inducible human embryonic kidney 293 cell lines for overexpression of FLAG-tagged Bα/δ regulatory subunits by ∼3-fold or knock-down of Bα by greater than 70% compared with endogenous levels. The expression of functional epitope-tagged B subunits was confirmed by the detection of A and C subunits as well as Phosphatase activity in FLAG immune complexes from extracts of cells overexpressing the FLAG-Bα/δ subunit. Western analysis of the cell extracts using phosphospecific antibodies for MEK1/2 and ERK1/2 demonstrated that activation of these kinases in response to epidermal growth factor was markedly diminished in Bα knock-down cells but elevated in Bα- and Bδ-overexpressing cells as compared with control cells. In parallel with the activation of MEK1/2 and ERK1/2, the inhibitory phosphorylation site of Raf1 (Ser-259) was dephosphorylated in cells overexpressing Bα or Bδ. Pharmacological inhibitor studies as well as reporter assays for ERK-dependent activation of the transcription factor Elk1 revealed that the PP2A holoenzymes ABαC and ABδC act downstream of Ras and upstream of MEK1 to promote activation of this MAPK signaling cascade. Furthermore both PP2A holoenzymes were found to associate with Raf1 and catalyze dephosphorylation of inhibitory phospho-Ser-259. Together these findings indicate that PP2A ABαC and ABδC holoenzymes function as positive regulators of Raf1-MEK1/2-ERK1/2 signaling by targeting Raf1.
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Positive Regulation of IκB Kinase Signaling by Protein Serine/Threonine Phosphatase 2A
The Journal of biological chemistry, 2005Co-Authors: Arlene E. Kray, Robert S. Carter, Kevin N. Pennington, Rey J. Gomez, Laura E. Sanders, Joan M. Llanes, Wasif N. Khan, Dean W. Ballard, Brian E WadzinskiAbstract:Transcription factor NF-κB plays a key regulatory role in the cellular response to pro-inflammatory cytokines such as tumor necrosis factor-α (TNF). In the absence of TNF, NF-κB is sequestered in the cytoplasm by inhibitory IκB Proteins. Phosphorylation of IκBby the β-catalytic subunit of IKK, a multicomponent IκB kinase, targets the inhibitor for proteolytic destruction and facilitates nuclear translocation of NF-κB. This pathway is initiated by TNF-dependent phosphorylation of T loop serines in IKKβ, which greatly stimulates IκB kinase activity. Prior in vitro mixing experiments indicate that Protein serine/threonine Phosphatase 2A (PP2A) can dephosphorylate these T loop serines and inactivate IKK, suggesting a negative regulatory role for PP2A in IKK signaling. Here we provided several in vivo lines of evidence indicating that PP2A plays a positive rather than a negative role in the regulation of IKK. First, TNF-induced degradation of IκB is attenuated in cells treated with okadaic acid or fostriecin, two potent inhibitors of PP2A. Second, PP2A forms stable complexes with IKK in untransfected mammalian cells. This interaction is critically dependent on amino acid residues 121–179 of the IKKγ regulatory subunit. Third, deletion of the PP2A-binding site in IKKγ attenuates T loop phosphorylation and catalytic activation of IKKβ in cells treated with TNF. Taken together, these data provide strong evidence that the formation of IKK·PP2A complexes is required for the proper induction of IκB kinase activity in vivo.
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histone deacetylase 3 hdac3 activity is regulated by interaction with Protein serine threonine Phosphatase 4
Genes & Development, 2005Co-Authors: Xiaohong Zhang, Brian E Wadzinski, Yukiyasu Ozawa, Edward SetoAbstract:Histone deacetylase 3 (HDAC3) is one of four members of the human class I HDACs that regulates gene expression by deacetylation of histones and nonhistone Proteins. Early studies have suggested that HDAC3 activity is regulated by association with the corepressors N-CoR and SMRT. Here we demonstrate that, in addition to Protein–Protein interactions with NCoR/SMRT, the activity of HDAC3 is regulated by both phosphorylation and dephosphorylation. A Protein kinase CK2 phosphoacceptor site in the HDAC3 Protein was identified at position Ser424, which is a nonconserved residue among the class I HDACs. Mutation of this residue was found to reduce deacetylase activity. Interestingly, unlike other class I HDACs, HDAC3 uniquely copurifies with the catalytic and regulatory subunits of the Protein serine/threonine Phosphatase 4 complex (PP4c/PP4R1). Furthermore, HDAC3 complexes displayed Protein Phosphatase activity and a series of subsequent mutational analyses revealed that the N terminus of HDAC3 (residues 1–122) was both necessary and sufficient for HDAC3–PP4c interactions. Significantly, both overexpression and siRNA knock-down approaches, and analysis of cells devoid of PP4c, unequivocally show that HDAC3 activity is inversely proportional to the cellular abundance of PP4c. These findings therefore further highlight the importance of Protein–Protein interactions and extend the significance of dephosphorylation in the regulation of HDAC activity, as well as present a novel alternative pathway by which HDAC3 activity is regulated.
X.-w. Zhang - One of the best experts on this subject based on the ideXlab platform.
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Protein Serine/Threonine Phosphotase-2A is Differentially Expressed and Regulates Eye Development in Vertebrates
Current molecular medicine, 2013Co-Authors: Wenbin Liu, X.-w. Zhang, Mi Deng, L. Nie, S.-s. Hui, W. Duan, Min Tao, Chun Zhang, Jinhui LiuAbstract:Protein serine/threonine Phosphatase-2A (PP-2A) is one of the key enzymes responsible for dephosphorylation in vertebrates. PP-2A-mediated dephosphorylation participates in many different biological processes including cell proliferation, differentiation, transformation, apoptosis, autophage and senescence. However, whether PP-2A directly controls animal development remains to be explored. Here, we present direct evidence to show that PP-2A displays important functions in regulating eye development of vertebrates. Using goldfish as a model system, we have demonstrated the following novel information. First, inhibition of PP-2A activity leads to significant death of the treated embryos, which is derived from blastomere apoptosis associated with enhanced phosphorylation of Bcl-XL at Ser-62, and the survived embryos displayed severe phenotype in the eye. Second, knockdown of PP-2A with morpholino oligomers leads to significant death of the injected embryos. The survived embryos from PP-2A knockdown displayed clear retardation in lens differentiation. Finally, overexpression of each catalytic subunit of PP-2A also causes death of majority of the injected embryos and leads to absence of goldfish eye lens or severely disturbed differentiation. Together, our results provide direct evidence that Protein Phosphatase-2A is important for normal eye development in goldfish.
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Protein Serine/Threonine Phosphotase-1 is Essential in Governing Normal Development of Vertebrate Eye
Current molecular medicine, 2012Co-Authors: Wenbin Liu, L. Nie, Q. Yan, Fangyuan Liu, Xiangcheng Tang, H.-g. Chen, Jin Ping Liu, X.-w. ZhangAbstract:Protein serine/threonine Phosphatase-1 (PP-1) is one of the key enzymes responsible for dephosphorylation in vertebrates. Protein dephosphorylation via PP-1 is implicated in many different biological processes including gene expression, cell cycle control, transformation, neuronal transmission, apoptosis, autophage and senescence. However, whether PP-1 directly controls animal development remains to be investigated. Here, we present direct evidence to show that PP-1 plays an essential role in regulating eye development of vertebrates. Using goldfish as a model system, we have shown the following novel results. First, inhibition of PP-1 activity leads to death of a majority of the treated embryos, and the survived embryos displayed severe phenotype in the eye. Second, knockdown of each catalytic subunit of PP-1 with morpholino oligomers leads to partial (PP-lα knockdown) or complete (PP-lβ or PP-lγ knockdown) death of the injected embryos. The survived embryos from PP-1α knockdown displayed clear retardation in lens differentiation. Finally, overexpression of each subunit of PP-1 also causes death of majority of the injected embryos and leads to abnormal development of goldfish eye. Mechanistically, Pax-6 is one of the major downstream targets mediating the effects of PP-1 function since the eye phenotype in Pax-6 knockdown fish is similar to that derived from overexpression of PP-1. Together, our results for the first time provide direct evidence that Protein Phosphatase-1 plays a key role in governing normal eye formation during goldfish development.
Wenbin Liu - One of the best experts on this subject based on the ideXlab platform.
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Protein Serine/Threonine Phosphotase-2A is Differentially Expressed and Regulates Eye Development in Vertebrates
Current molecular medicine, 2013Co-Authors: Wenbin Liu, X.-w. Zhang, Mi Deng, L. Nie, S.-s. Hui, W. Duan, Min Tao, Chun Zhang, Jinhui LiuAbstract:Protein serine/threonine Phosphatase-2A (PP-2A) is one of the key enzymes responsible for dephosphorylation in vertebrates. PP-2A-mediated dephosphorylation participates in many different biological processes including cell proliferation, differentiation, transformation, apoptosis, autophage and senescence. However, whether PP-2A directly controls animal development remains to be explored. Here, we present direct evidence to show that PP-2A displays important functions in regulating eye development of vertebrates. Using goldfish as a model system, we have demonstrated the following novel information. First, inhibition of PP-2A activity leads to significant death of the treated embryos, which is derived from blastomere apoptosis associated with enhanced phosphorylation of Bcl-XL at Ser-62, and the survived embryos displayed severe phenotype in the eye. Second, knockdown of PP-2A with morpholino oligomers leads to significant death of the injected embryos. The survived embryos from PP-2A knockdown displayed clear retardation in lens differentiation. Finally, overexpression of each catalytic subunit of PP-2A also causes death of majority of the injected embryos and leads to absence of goldfish eye lens or severely disturbed differentiation. Together, our results provide direct evidence that Protein Phosphatase-2A is important for normal eye development in goldfish.
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Protein Serine/Threonine Phosphotase-1 is Essential in Governing Normal Development of Vertebrate Eye
Current molecular medicine, 2012Co-Authors: Wenbin Liu, L. Nie, Q. Yan, Fangyuan Liu, Xiangcheng Tang, H.-g. Chen, Jin Ping Liu, X.-w. ZhangAbstract:Protein serine/threonine Phosphatase-1 (PP-1) is one of the key enzymes responsible for dephosphorylation in vertebrates. Protein dephosphorylation via PP-1 is implicated in many different biological processes including gene expression, cell cycle control, transformation, neuronal transmission, apoptosis, autophage and senescence. However, whether PP-1 directly controls animal development remains to be investigated. Here, we present direct evidence to show that PP-1 plays an essential role in regulating eye development of vertebrates. Using goldfish as a model system, we have shown the following novel results. First, inhibition of PP-1 activity leads to death of a majority of the treated embryos, and the survived embryos displayed severe phenotype in the eye. Second, knockdown of each catalytic subunit of PP-1 with morpholino oligomers leads to partial (PP-lα knockdown) or complete (PP-lβ or PP-lγ knockdown) death of the injected embryos. The survived embryos from PP-1α knockdown displayed clear retardation in lens differentiation. Finally, overexpression of each subunit of PP-1 also causes death of majority of the injected embryos and leads to abnormal development of goldfish eye. Mechanistically, Pax-6 is one of the major downstream targets mediating the effects of PP-1 function since the eye phenotype in Pax-6 knockdown fish is similar to that derived from overexpression of PP-1. Together, our results for the first time provide direct evidence that Protein Phosphatase-1 plays a key role in governing normal eye formation during goldfish development.
P T Cohen - One of the best experts on this subject based on the ideXlab platform.
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Drosophila PPY, a novel male specific Protein serine/threonine Phosphatase localised in somatic cells of the testis.
Journal of cell science, 1995Co-Authors: C G Armstrong, D J Mann, N Berndt, P T CohenAbstract:Drosophila Protein Phosphatase Y (PPY) displays 64% amino acid identity to Protein serine/threonine Phosphatase 1 (PP1) and 39% to Protein Phosphatase 2A (PP2A). Here we show by expression of cDNA in bacteria, that PPY is a Protein serine Phosphatase and that its biochemical properties are distinct from PP1 in both substrate specificity and regulation by the thermostable inhibitory Proteins inhibitor 1 and inhibitor 2. We also demonstrate that PPY is a novel testis specific Protein Phosphatase by analysis of both mRNA and Protein distribution. More precise immunolocalisation within the testis, using affinity purified anti-PPY Protein and anti-PPY peptide antibodies, shows that PPY is present in somatic cyst cells, which encase the germ cells. The predominant location of PPY is in the nuclei of both head and tail cyst cells throughout the length of the testis except for the apical tip. The distribution of PPY, coupled with its unique biochemical properties, suggests that PPY may be required to prevent cyst cell division, increase transcription for provision of nutrients to the germ cells and/or provide a signal for spermatocyte differentiation.
