B55alpha PP2A holoenzymes modulate the phosphorylation status of the retinoblastoma-related protein p107 and its activation.

Pocket proteins negatively regulate transcription of E2F-dependent genes and progression through the G(0)/G(1) transition and the cell cycle restriction point in G(1). Pocket protein repressor activities are inactivated via phosphorylation at multiple Pro-directed Ser/Thr sites by the coordinated action of G(1) and G(1)/S cyclin-dependent kinases. These phosphorylations are reversed by the action of two families of Ser/Thr phosphatases: PP1, which has been implicated in abrupt dephosphorylation of retinoblastoma protein (pRB) in mitosis, and PP2A, which plays a role in an equilibrium that counteracts cyclin-dependent kinase (CDK) action throughout the cell cycle. However, the identity of the trimeric PP2A holoenzyme(s) functioning in this process is unknown. Here we report the identification of a PP2A trimeric holoenzyme containing B55α, which plays a major role in restricting the phosphorylation state of p107 and inducing its activation in human cells. Our data also suggest targeted selectivity in the interaction of pocket proteins with distinct PP2A holoenzymes, which is likely necessary for simultaneous pocket protein activation.

PP2A is required for centromeric localization of Sgo1 and proper chromosome segregation.

Loss of sister-chromatid cohesion triggers chromosome segregation in mitosis and occurs through two mechanisms in vertebrate cells: (1) phosphorylation and removal of cohesin from chromosome arms by mitotic kinases, including Plk1, during prophase, and (2) cleavage of centromeric cohesin by separase at the metaphase-anaphase transition. Bub1 and the MEI-S332/Shugoshin (Sgo1) family of proteins protect centromeric cohesin from mitotic kinases during prophase. We show that human Sgo1 binds to protein phosphatase 2A (PP2A). PP2A localizes to centromeres in a Bub1-dependent manner. The Sgo1-PP2A interaction is required for centromeric localization of Sgo1 and proper chromosome segregation in human cells. Depletion of Plk1 by RNA interference (RNAi) restores centromeric localization of Sgo1 and prevents chromosome missegregation in cells depleted of PP2A_Aalpha. Our findings suggest that Bub1 targets PP2A to centromeres, which in turn maintains Sgo1 at centromeres by counteracting Plk1-mediated chromosome removal of Sgo1.

Acute regulation of renal Na+/H+ exchanger NHE3 by dopamine: role of protein phosphatase 2A.

Nephrogenic dopamine is a potent natriuretic paracrine/autocrine hormone that is central for mammalian sodium homeostasis. In the renal proximal tubule, dopamine induces natriuresis partly via inhibition of the sodium/proton exchanger NHE3. The signal transduction pathways and mechanisms by which dopamine inhibits NHE3 are complex and incompletely understood. This manuscript describes the role of the serine/threonine protein phosphatase 2A (PP2A) in the regulation of NHE3 by dopamine. The PP2A regulatory subunit B56δ (coded by the Ppp2r5d gene) directly associates with more than one region of the carboxy-terminal hydrophilic putative cytoplasmic domain of NHE3 (NHE3-cyto), as demonstrated by yeast-two-hybrid, coimmunoprecipitation, blot overlay, and in vitro pull-down assays. Phosphorylated NHE3-cyto is a substrate for purified PP2A in an in vitro dephosphorylation reaction. In cultured renal cells, inhibition of PP2A by either okadaic acid or by overexpression of the simian virus 40 (SV40) small T antigen blocks the ability of dopamine to inhibit NHE3 activity and to reduce surface NHE3 protein. Dopamine-induced NHE3 redistribution is also blocked by okadaic acid ex vivo in rat kidney cortical slices. These studies demonstrate that PP2A is an integral and critical participant in the signal transduction pathway between dopamine receptor activation and NHE3 inhibition.

The use of RNA interference to analyze protein phosphatase function in mammalian cells.

The use of RNA interference to knock down protein phosphatases has proven to be a valuable approach to understanding the functions of these enzymes in mammalian cells. Many protein phosphatases exist as multisubunit and multigene families, which has made it difficult to assess their physiological functions using traditional approaches. The ability to selectively knock down specific subunits and individual isoforms with RNA interference has begun to make it possible to determine the contributions of individual phosphatase proteins to cellular signaling. This chapter describes methods for knocking down protein phosphatases with small interfering RNAs in easily transfectable cells and by the introduction of short-hairpin RNAs into less tractable cells using lentivirus vectors.

PR55α Subunit of Protein Phosphatase 2A Supports the Tumorigenic and Metastatic Potential of Pancreatic Cancer Cells by Sustaining Hyperactive Oncogenic Signaling.

The protein phosphatase 2 (PP2A) holoenzyme consists of a catalytic subunit, a scaffold subunit, and a regulatory subunit. Based on loss-of-function analysis using PP2A catalytic inhibitors or inhibition via tumor viral antigens, limited studies suggest that PP2A is a putative tumor suppressor. However, PP2A has also been shown to facilitate the activation of oncogenic signaling pathways when associated with specific regulatory subunits. In this study, we investigated the possible oncogenic role of PP2A in pancreatic cancer. We found a striking increase in the expression of PR55α (PPP2R2A), a PP2A regulatory subunit, in pancreatic cancer cells compared with normal pancreatic epithelial cells. Consistently, PR55α expression was markedly elevated in pancreatic ductal adenocarcinoma tissues compared with adjacent normal pancreatic tissues (P < 0.0001) and correlated with poor survival of pancreatic cancer patients (P < 0.0003). RNAi-mediated depletion of PR55α in pancreatic cancer cell lines resulted in diminished phosphorylation of both AKT and ERK1/2 (MAPK3/1) and decreased protein levels of ß-catenin (CTNNB1). Accordingly, pancreatic cancer cells with reduced PR55α expression exhibited significantly impaired properties of transformation, including attenuated cell growth, clonogenicity, mobility, and anchorage-independent growth. Moreover, orthotopic implantation of PR55α-depleted pancreatic cancer cells into nude mice resulted in markedly reduced tumorigenicity (P < 0.001) and distant metastases. Together, these results suggest that PR55α promotes pancreatic cancer development by sustaining hyperactivity of multiple oncogenic signaling pathways, including AKT, ERK, and Wnt. These studies also provide a basis for exploring PR55α as a diagnostic or therapeutic target in pancreatic cancer. Cancer Res; 76(8); 2243-53. ©2016 AACR.

Genetic modulation of tau phosphorylation in the mouse.

The axonal microtubule stabilizing protein tau is hyperphosphorylated in several neurodegenerative conditions, including Alzheimer's disease, yet the genes that regulate tau phosphorylation are largely unknown. Disabled-1 (Dab1) is a cytoplasmic adapter protein that interacts with apolipoprotein E (ApoE) receptors and controls neuronal positioning during embryonic brain development. We have investigated the role of Dab1 in tau phosphorylation. We found that wild-type Dab1, but not a mutant lacking tyrosine phosphorylation sites, protects mice from the hyperphosphorylation of tau. However, the absence of Dab1 is not sufficient to cause tau hyperphosphorylation, because hyperphosphorylation is manifested only when Dab1 is mutated in specific mouse strain backgrounds. Tau hyperphosphorylation correlates with early death in susceptible mouse strains, and it occurs in the neurons of the hippocampus and dentate gyrus. By quantitative trait locus (QTL) analysis of Dab1-deficient mice on a hybrid strain background, we uncovered one significant and three suggestive chromosomal loci that modulate tau phosphorylation. Two of these QTL regions contain genes that are defective in early onset Alzheimer's disease. Our findings suggest that Dab1 gene disruption sensitizes mice to tau hyperphosphorylation contingent on specific haplotypes that are linked to Alzheimer's disease loci. Dab1 mutant mice provide an animal model for studying the relationships between ApoE receptors, tau hyperphosphorylation, and Alzheimer's disease.

Analysis of protein phosphatase function in Drosophila cells using RNA interference.

Double stranded RNA-mediated RNA interference is an effective method to downregulate the levels of protein phosphatases in Drosophila S2 cells. In many cases, nearly complete ablation of the targeted protein can be achieved. RNAi-mediated knockdown of protein phosphatases is akin to pharmacological inhibition with drugs and can be used to determine the roles of specific protein phosphatases in intact cells. RNAi can avoid the problems associated with less than adequate specificity of phosphatase inhibitors. Although information about the signaling pathways present in Drosophila S2 cells is not as well developed as many mammalian cell lines, the Drosophila system is particularly attractive for the study of oligomeric phosphatases like PP2A. Drosophila has far fewer isoforms for the phosphatases we have examined. This is especially true of the genes for PP2A regulatory subunits where over 50 isoforms are present in mammals but only four are present in Drosophila. Once hypotheses regarding phosphatase function have been generated from RNAi experiments in S2 cells, they can potentially be tested utilizing recent advances in the use of siRNAs to conduct RNAi experiments in mammalian cell lines. RNAi in Drosophila S2 cells has proven to be a powerful technique for identifying physiological functions of signaling proteins. The RNAi method is straightforward and works routinely with almost all proteins. RNAi in S2 cells can be used to assess the role of signaling proteins in specific pathways and as a screening tool to identify new roles for signaling molecules. For example, results from RNAi analysis of PP2A show that regulation of MAP kinase signaling involves the R2/B regulatory subunit and that the R5/B56 subunits play a previously unidentified role in apoptosis. While RNAi in Drosophila S2 cells is a powerful tool for analyzing protein function, the method does have limitations. Foremost, cells may exhibit an RNAi response to any nonspecific dsRNA, even in the absence of interferon. Therefore, physiological processes that respond to nonspecific dsRNA will be difficult to study. A second limitation is the need to produce antibodies that react with Drosophila isoforms. We have found that many antibodies to mammalian protein phosphatases do not cross-react with the corresponding Drosophila proteins. Finally, the physiology and signaling pathways of S2 cells have not been extensively studied. This lack of information limits the number of available readouts that can be used when assessing the effects of protein knockdowns.

Structure of the Ca2+-dependent PP2A heterotrimer and insights into Cdc6 dephosphorylation.

The B″/PR72 family of protein phosphatase 2A (PP2A) is an important PP2A family involved in diverse cellular processes, and uniquely regulated by calcium binding to the regulatory subunit. The PR70 subunit in this family interacts with cell division control 6 (Cdc6), a cell cycle regulator important for control of DNA replication. Here, we report crystal structures of the isolated PR72 and the trimeric PR70 holoenzyme at a resolution of 2.1 and 2.4 Å, respectively, and in vitro characterization of Cdc6 dephosphorylation. The holoenzyme structure reveals that one of the PR70 calcium-binding motifs directly contacts the scaffold subunit, resulting in the most compact scaffold subunit conformation among all PP2A holoenzymes. PR70 also binds distinctively to the catalytic subunit near the active site, which is required for PR70 to enhance phosphatase activity toward Cdc6. Our studies provide a structural basis for unique regulation of B″/PR72 holoenzymes by calcium ions, and suggest the mechanisms for precise control of substrate specificity among PP2A holoenzymes.

Protein phosphatase 2A is targeted to cell division control protein 6 by a calcium-binding regulatory subunit.

The cell division control protein 6 (Cdc6) is essential for formation of pre-replication complexes at origins of DNA replication. Phosphorylation of Cdc6 by cyclin-dependent kinases inhibits ubiquitination of Cdc6 by APC/C(cdh1) and degradation by the proteasome. Experiments described here show that the PR70 member of the PPP2R3 family of regulatory subunits targets protein phosphatase 2A (PP2A) to Cdc6. Interaction with Cdc6 is mediated by residues within the C terminus of PR70, whereas interaction with PP2A requires N-terminal sequences conserved within the PPP2R3 family. Two functional EF-hand calcium-binding motifs mediate a calcium-enhanced interaction of PR70 with PP2A. Calcium has no effect on the interaction of PR70 with Cdc6 but enhances the association of PP2A with Cdc6 through its effects on PR70. Knockdown of PR70 by RNA interference results in an accumulation of endogenous and expressed Cdc6 protein that is dependent on the cyclin-dependent protein kinase phosphorylation sites on Cdc6. Knockdown of PR70 also causes G(1) arrest, suggesting that PR70 function is critical for progression into S phase. These observations indicate that PP2A can be targeted in a calcium-regulated manner to Cdc6 via the PR70 subunit, where it plays a role in regulating protein phosphorylation and stability.

PP2A: unveiling a reluctant tumor suppressor.

Although evidence has suggested that the serine/threonine protein phosphatase 2A (PP2A) might be a tumor suppressor protein, it has been difficult to pin down its role in tumor suppression because it acts in a wide variety of signaling pathways. Recent findings, including work in this issue by Junttila et al. (2007), provide convincing evidence that suppression of PP2A activity cooperates with other oncogenic changes to cause transformation of multiple cell types.

The 3D structure of protein phosphatase 2A: new insights into a ubiquitous regulator of cell signaling.

Protein phosphatase 2A (PP2A) is a serine/threonine phosphatase implicated in cancer. Three new crystal structures of PP2A show how it interacts with inhibitory toxins and with one of its regulatory subunits. The structures also explain how specific site mutations may lead to cancer and suggest a novel role for PP2A methylation in the formation of PP2A holoenzymes.

Functional analysis of the PP2A subfamily of protein phosphatases in regulating Drosophila S6 kinase.

Phosphorylation and activation of ribosomal S6 protein kinase is an important link in the regulation of cell size by the target of rapamycin (TOR) protein kinase. A combination of selective inhibition and RNA interference were used to test the roles of members of the PP2A subfamily of protein phosphatases in dephosphorylation of Drosophila S6 kinase (dS6K). Treatment of Drosophila Schneider 2 cells with calyculin A, a selective inhibitor of PP2A-like phosphatases, resulted in a 7-fold increase in the basal level of dS6K phosphorylation at the TOR phosphorylation site (Thr398) and blocked dephosphorylation following inactivation of TOR by amino acid starvation or rapamycin treatment. Knockdown of the PP2A catalytic subunit increased basal dS6K phosphorylation and inhibited dephosphorylation induced by amino acid withdrawal. In contrast, depletion of the catalytic subunits of the other two members of the subfamily did not enhance dS6K phosphorylation. Knockdown of PP4 caused a 20% decrease in dS6K phosphorylation and knockdown of PP6 had no effect. Knockdown of the Drosophila B56-2 subunit resulted in enhanced dephosphorylation of dS6K following removal of amino acids. In contrast, knockdown of the homologs of the other PP2A regulatory subunits had no effects. Knockdown of the Drosophila homolog of the PP2A/PP4/PP6 interaction protein alpha4/Tap42 did not affect S6K phosphorylation, but did induce apoptosis. These results indicate that PP2A, but not other members of this subfamily, is likely to be a major S6K phosphatase in intact cells and is consistent with an important role for this phosphatase in the TOR pathway.

Mitochondrial protein phosphatase 2A regulates cell death induced by simulated ischemia in kidney NRK-52E cells.

Acute renal failure can occur after an ischemic injury and results in significant mortality. The stress-signaling pathways that are activated during renal ischemia are unknown. PP2A has emerged as an important regulator of cell death. To study the role of PP2A in ischemia-induced cell death, we used an in vitro model of simulated ischemia. In the present study, simulated ischemia in rat renal tubule epithelial NRK-52E cells (A) results in cell death that involves both necrosis and apoptosis, (B) activates PP2A, and (C) up-regulates the PP2A B56 alpha regulatory subunit. Previous data have shown that PKC alpha negatively regulates B56 alpha protein expression. Consistent with this finding, simulated ischemia suppressed PKC alpha and up-regulated B56 alpha. Treatment of NRK-52E cells with ceramide suppressed PKC alpha and activated PP2A in a manner that mimicked simulated ischemia. A role for PP2A in simulated ischemia-induced cell death is likely since inhibition of PP2A protected NRK-52E cells. In addition, overexpression of exogenous B56 alpha but not B55 in NRK-52E cells enhanced simulated ischemia-induced cell death. These findings suggest that activation of a PP2A isoform that contains the B56 alpha regulatory subunit is required for ischemia-induced cell death in kidney epithelial proximal tubule cells.

A functional genomics analysis of the B56 isoforms of Drosophila protein phosphatase 2A.

Members of the B56 family of protein phosphatase 2A (PP2A) regulatory subunits play crucial roles in Drosophila cell survival. Distinct functions of two B56 subunits were investigated using a combination of RNA interference, DNA microarrays, and proteomics. RNA interference-mediated knockdown of the B56-1 subunit (PP2A-B') but not the catalytic (mts) or B56-2 subunit (wdb) of PP2A resulted in increased expression of the apoptotic inducers reaper and sickle. Co-knockdown of B56-1 with reaper, but not with sickle, reduced the apoptosis caused by depletion of the B56 subunits. Two-dimensional gel electrophoresis and mass spectrometry identified proteins modified in cells depleted of PP2A subunits. These included generation of caspase-dependent cleavage products, increases in protein abundance, and covalent modifications. Results suggested that up-regulation of the ribosome-associated protein stubarista can serve as a sensitive marker of apoptosis. Up-regulation of transcripts for multiple glutathione transferases and other proteins suggested that loss of PP2A affected pathways involved in the response to oxidative stress. Knockdown of PP2A elevated basal JNK activity and substantially decreased activation of ERK in response to oxidative stress. The results reveal that the B56-containing isoform of PP2A functions within multiple signaling pathways, including those that regulate expression of reaper and the response to oxidative stress, thus promoting cell survival in Drosophila.

The glycine 90 to aspartate alteration in the Abeta subunit of PP2A (PPP2R1B) associates with breast cancer and causes a deficit in protein function.

Mutations of the PPP2R1B gene, which encodes the Abeta scaffolding subunit of serine/threonine protein phosphatase 2A (PP2A), have been identified in several types of cancer including lung and breast carcinoma. One of these mutations results in an alteration of glycine 90 to aspartic acid (G90D), which has been found in both tumor and genomic DNA, raising the possibility that it is associated with an increased risk for cancer. A novel microarray-based technology was used to screen for this single-nucleotide polymorphism in 387 cancer patients and 329 control individuals. These data were used for case-control and family-based comparisons in order to study the association of this polymorphism with susceptibility to lung carcinoma, breast carcinoma, and acute lymphoblastic leukemia. The frequency of the G90D polymorphism in breast cancer patients was significantly higher in cases (3%) than in controls (0.3%). The wild-type Abeta subunit interacted with the B56gamma (PPP2R5C), PR72 (PPP2R3A), and PR48 subunits of PP2A but did not interact with the B55alpha (PPP2R2A), B56alpha (PPP2R5A), or B56beta (PPP2R5B) regulatory subunits in an in vitro binding assay. The G90D alteration inhibited the interaction of Abeta with the B56gamma subunit but had no effect on binding to the PR72 subunit. These results provide evidence that the G90D alteration of the Abeta subunit of PP2A is associated with a low frequency of breast carcinoma and that the role of this alteration in transformation is likely to involve decreased interaction with the B56gamma regulatory subunit.

The gatekeeper residue controls autoactivation of ERK2 via a pathway of intramolecular connectivity.

Studies of protein kinases have identified a "gatekeeper" residue, which confers selectivity for binding nucleotides and small-molecule inhibitors. We report that, in the MAP kinase ERK2, mutations at the gatekeeper residue unexpectedly lead to autoactivation due to enhanced autophosphorylation of regulatory Tyr and Thr sites within the activation lip that control kinase activity. This occurs through an intramolecular mechanism, indicating that the gatekeeper residue indirectly constrains flexibility at the activation lip, precluding access of the phosphoacceptor residues to the catalytic base. Other residues that interact with the gatekeeper site to form a hydrophobic cluster in the N-terminal domain also cause autoactivation when mutated. Hydrogen-exchange studies of a mutant within this cluster reveal perturbations in the conserved DFG motif, predicting a route for side chain connectivity from the hydrophobic cluster to the activation lip. Mutations of residues along this route support this model, explaining how information about the gatekeeper residue identity can be transmitted to the activation lip. Thus, an N-terminal hydrophobic cluster that includes the gatekeeper forms a novel structural unit, which functions to maintain the "off" state of ERK2 before cell signal activation.

Phosphoproteomics: new insights into cellular signaling.

Developments in the field of phosphoproteomics have been fueled by the need simultaneously to monitor many different phosphoproteins within the signaling networks that coordinate responses to changes in the cellular environment. This article presents a brief review of phosphoproteomics with an emphasis on the biological insights that have been derived so far.

InsPecT: identification of posttranslationally modified peptides from tandem mass spectra.

Reliable identification of posttranslational modifications is key to understanding various cellular regulatory processes. We describe a tool, InsPecT, to identify posttranslational modifications using tandem mass spectrometry data. InsPecT constructs database filters that proved to be very successful in genomics searches. Given an MS/MS spectrum S and a database D, a database filter selects a small fraction of database D that is guaranteed (with high probability) to contain a peptide that produced S. InsPecT uses peptide sequence tags as efficient filters that reduce the size of the database by a few orders of magnitude while retaining the correct peptide with very high probability. In addition to filtering, InsPecT also uses novel algorithms for scoring and validating in the presence of modifications, without explicit enumeration of all variants. InsPecT identifies modified peptides with better or equivalent accuracy than other database search tools while being 2 orders of magnitude faster than SEQUEST, and substantially faster than X!TANDEM on complex mixtures. The tool was used to identify a number of novel modifications in different data sets, including many phosphopeptides in data provided by Alliance for Cellular Signaling that were missed by other tools.

A functional role for the B56 alpha-subunit of protein phosphatase 2A in ceramide-mediated regulation of Bcl2 phosphorylation status and function.

Recently it has been shown that the potent apoptotic agent ceramide activates a mitochondrial protein phosphatase 2A (PP2A) and promotes dephosphorylation of the anti-apoptotic molecule Bcl2 (Ruvolo, P. P., Deng, X., Ito, T., Carr, B. K., and May, W. S. (1999) J. Biol. Chem. 274, 20296-20300). In cells expressing Bcl2, dephosphorylation of Bcl2 appears to be required for ceramide-induced cell death because treatment of cells with low doses of the PP2A inhibitor okadaic acid blocks Bcl2 dephosphorylation and promotes cell survival. Furthermore, the non-phosphorylatable (i.e. PP2A-resistant) gain-of-function S70E mutant Bcl2 can protect cells from ceramide-induced apoptosis. These findings support a model whereby Bcl2 function is regulated by PP2A. PP2A is a heterotrimer that contains a catalytic C-subunit, a structural A-subunit, and a regulatory B-subunit. The A- and C-subunits are fairly conserved and ubiquitously expressed, and they form the catalytic complex of the phosphatase. In contrast, there are at least three families of diverse B-subunit molecules that vary in expression temporally and by tissue type. It is hypothesized that ceramide regulates PP2A via the B-subunit. Thus, understanding the mechanism of how PP2A regulates Bcl2 phosphorylation status and how ceramide might regulate this process requires identification of the regulatory B-subunit of PP2A that comprises the Bcl2 phosphatase. Results indicate that the B56 alpha-subunit is a candidate regulatory subunit of the physiologic Bcl2 phosphatase since (a) B56 alpha associates with Bcl2 as evidenced by pull-down experiments, (b) B56 alpha co-localizes with Bcl2 in mitochondrial membranes, (c) ceramide promotes translocation of B56 alpha to mitochondrial membranes, and (d) overexpression of B56 alpha promotes mitochondrial PP2A activity and Bcl2 dephosphorylation and potentiates cell killing with ceramide. These findings suggest a role for B56 alpha in regulating the Bcl2 phosphatase.

Actions of PP2A on the MAP kinase pathway and apoptosis are mediated by distinct regulatory subunits.

Individual subunits of protein phosphatase 2A (PP2A), protein phosphatase 4, and protein phosphatase 5 were knocked out in Drosophila Schneider 2 cells by using RNA interference. Ablation of either the scaffold (A) or catalytic (C) subunits of PP2A caused the disappearance of all PP2A subunits. Treating cells with double-stranded RNA targeting all four of the Drosophila PP2A regulatory subunits caused the disappearance of both the A and C subunits. The loss of PP2A subunits was associated with decreased protein stability indicating that only the heterotrimeric forms of PP2A are stable in intact cells. Ablation of total PP2A by using double-stranded RNA against either the A or C subunit, or specific ablation of the R2/B regulatory subunit, enhanced insulin-induced ERK activation. These results indicated that the R2/B subunit targets PP2A to the mitogen-activated protein (MAP) kinase cascade in Schneider 2 cells, where it acts as a negative regulator. A severe loss of viability occurred in cells in which total PP2A or both isoforms of the Drosophila R5/B56 subunit had been ablated. The reduced viability of these cells correlated with the induction of markers of apoptosis including membrane blebbing and stimulation of caspase-3-like activity. These observations indicated that PP2A has a powerful antiapoptotic activity that is specifically mediated by the R5/B56 regulatory subunits. In contrast to PP2A, ablation of protein phosphatase 4 caused only a slight reduction in cell growth but had no effect on MAP kinase signaling or apoptosis. Depletion of protein phosphatase 5 had no effects on MAP kinase, cell growth, or apoptosis.