B56δ long-disordered arms form a dynamic PP2A regulation interface coupled with global allostery and Jordan's syndrome mutations.

Intrinsically disordered regions (IDR) and short linear motifs (SLiMs) play pivotal roles in the intricate signaling networks governed by phosphatases and kinases. B56δ (encoded by PPP2R5D) is a regulatory subunit of protein phosphatase 2A (PP2A) with long IDRs that harbor a substrate-mimicking SLiM and multiple phosphorylation sites. De novo missense mutations in PPP2R5D cause intellectual disabilities (ID), macrocephaly, Parkinsonism, and a broad range of neurological symptoms. Our single-particle cryo-EM structures of the PP2A-B56δ holoenzyme reveal that the long, disordered arms at the B56δ termini fold against each other and the holoenzyme core. This architecture suppresses both the phosphatase active site and the substrate-binding protein groove, thereby stabilizing the enzyme in a closed latent form with dual autoinhibition. The resulting interface spans over 190 Šand harbors unfavorable contacts, activation phosphorylation sites, and nearly all residues with ID-associated mutations. Our studies suggest that this dynamic interface is coupled to an allosteric network responsive to phosphorylation and altered globally by mutations. Furthermore, we found that ID mutations increase the holoenzyme activity and perturb the phosphorylation rates, and the severe variants significantly increase the mitotic duration and error rates compared to the normal variant.

Quantitative proteomics and phosphoproteomics of PP2A-PPP2R5D variants reveal deregulation of RPS6 phosphorylation via converging signaling cascades.

Genetic germline variants of PPP2R5D (encoding: phosphoprotein phosphatase 2 regulatory protein 5D) result in PPP2R5D-related disorder (Jordan's Syndrome), which is characterized by intellectual disability, hypotonia, seizures, macrocephaly, autism spectrum disorder, and delayed motor skill development. The disorder originates from de novo single nucleotide mutations, generating missense variants that act in a dominant manner. Pathogenic mutations altering 13 different amino acids have been identified, with the E198K variant accounting for ∼40% of reported cases. However, the generation of a heterozygous E198K variant cell line to study the molecular effects of the pathogenic mutation has been challenging. Here, we use CRISPR-PRIME genomic editing to introduce a transition (c.592G>A) in a single PPP2R5D allele in HEK293 cells, generating E198K-heterozygous lines to complement existing E420K variant lines. We generate global protein and phosphorylation profiles of WT, E198K, and E420K cell lines and find unique and shared changes between variants and WT cells in kinase- and phosphatase-controlled signaling cascades. We observed ribosomal protein S6 (RPS6) hyperphosphorylation as a shared signaling alteration, indicative of increased ribosomal protein S6-kinase activity. Treatment with rapamycin or an RPS6-kinase inhibitor (LY2584702) suppressed RPS6 phosphorylation in both, suggesting upstream activation of mTORC1/p70S6K. Intriguingly, our data suggests ERK-dependent activation of mTORC1 in both E198K and E420K variant cells, with additional AKT-mediated mTORC1 activation in the E420K variant. Thus, although upstream activation of mTORC1 differs between PPP2R5D-related disorder genotypes, inhibition of mTORC1 or RPS6 kinases warrants further investigation as potential therapeutic strategies for patients.

Quantum-based modeling implies that bidentate Arg89-substrate binding enhances serine/threonine protein phosphatase-2A(PPP2R5D/PPP2R1A/PPP2CA)-mediated dephosphorylation.

PP2A-serine/threonine protein phosphatases function as heterotrimeric holoenzymes, composed of a common scaffold (A-subunit encoded by PPP2R1A/PPP2R1B), a common catalytic (C-subunit encoded by PPP2CA/PPP2CB), and one of many variable regulatory (B) subunits. The site of phosphoprotein phosphatase (PPP) hydrolysis features a bimetal system (M1/M2), an associated bridge hydroxide [W1(OH-)], and a highly-conserved core sequence. In the presumptive common mechanism, the phosphoprotein's seryl/threonyl phosphate coordinates the M1/M2 system, W1(OH-) attacks the central P atom, rupturing the antipodal bond, and simultaneously, a histidine/aspartate tandem protonates the exiting seryl/threonyl alkoxide. Based on studies of PPP5C, a conserved arginine proximal to M1 is also expected to bind the substrate's phosphate group in a bidentate fashion. However, in PP2A isozymes, the role of the arginine (Arg89) in hydrolysis is not clear because two independent structures for PP2A(PPP2R5C) and PP2A(PPP2R5D) show that Arg89 engages in a weak salt bridge at the B:C interface. These observations raise the question of whether hydrolysis proceeds with or without direct involvement of Arg89. The interaction of Arg89 with B:Glu198 in PP2A(PPP2R5D) is significant because the pathogenic E198K variant of B56δ is associated with irregular protein phosphorylation levels and consequent developmental disorders (Jordan's Syndrome; OMIM #616355). In this study, we perform quantum-based hybrid [ONIOM(UB3LYP/6-31G(d):UPM7)] calculations on 39-residue models of the PP2A(PPP2R5D)/pSer (phosphoserine) system to estimate activation barriers for hydrolysis in the presence of bidentate Arg89-substrate binding and when Arg89 is otherwise engaged in the salt-bridge interaction. Our solvation-corrected results yield ΔH‡ ≈ ΔE‡ = +15.5 kcal/mol for the former case, versus +18.8 kcal/mol for the latter, indicating that bidentate Arg89-substrate binding is critical for optimal catalytic function of the enzyme. We speculate that PP2A(PPP2R5D) activity is suppressed by B:Glu198 sequestration of C:Arg89 under native conditions, whereas the PP2A(PPP2R5D)-holoenzyme containing the E198K variant has a positively-charged lysine in this position that alters normal function.

FAK Activation Promotes SMC Dedifferentiation via Increased DNA Methylation in Contractile Genes.

RATIONALE: Vascular smooth muscle cells (SMCs) exhibit remarkable plasticity and can undergo dedifferentiation upon pathological stimuli associated with disease and interventions. OBJECTIVE: Although epigenetic changes are critical in SMC phenotype switching, a fundamental regulator that governs the epigenetic machineries regulating the fate of SMC phenotype has not been elucidated. METHODS AND RESULTS: Using SMCs, mouse models, and human atherosclerosis specimens, we found that FAK (focal adhesion kinase) activation elicits SMC dedifferentiation by stabilizing DNMT3A (DNA methyltransferase 3A). FAK in SMCs is activated in the cytoplasm upon serum stimulation in vitro or vessel injury and active FAK prevents DNMT3A from nuclear FAK-mediated degradation. However, pharmacological or genetic FAK catalytic inhibition forced FAK nuclear localization, which reduced DNMT3A protein via enhanced ubiquitination and proteasomal degradation. Reduced DNMT3A protein led to DNA hypomethylation in contractile gene promoters, which increased SMC contractile protein expression. RNA-sequencing identified SMC contractile genes as a foremost upregulated group by FAK inhibition from injured femoral artery samples compared with vehicle group. DNMT3A knockdown in injured arteries reduced DNA methylation and enhanced contractile gene expression supports the notion that nuclear FAK-mediated DNMT3A degradation via E3 ligase TRAF6 (TNF [tumor necrosis factor] receptor-associated factor 6) drives differentiation of SMCs. Furthermore, we observed that SMCs of human atherosclerotic lesions exhibited decreased nuclear FAK, which was associated with increased DNMT3A levels and decreased contractile gene expression. CONCLUSIONS: This study reveals that nuclear FAK induced by FAK catalytic inhibition specifically suppresses DNMT3A expression in injured vessels resulting in maintaining SMC differentiation by promoting the contractile gene expression. Thus, FAK inhibitors may provide a new treatment option to block SMC phenotypic switching during vascular remodeling and atherosclerosis.

Phosphatase inhibition by LB-100 enhances BMN-111 stimulation of bone growth.

Activating mutations in fibroblast growth factor receptor 3 (FGFR3) and inactivating mutations in the natriuretic peptide receptor 2 (NPR2) guanylyl cyclase both result in decreased production of cyclic GMP in chondrocytes and severe short stature, causing achondroplasia (ACH) and acromesomelic dysplasia, type Maroteaux, respectively. Previously, we showed that an NPR2 agonist BMN-111 (vosoritide) increases bone growth in mice mimicking ACH (Fgfr3Y367C/+). Here, because FGFR3 signaling decreases NPR2 activity by dephosphorylating the NPR2 protein, we tested whether a phosphatase inhibitor (LB-100) could enhance BMN-111-stimulated bone growth in ACH. Measurements of cGMP production in chondrocytes of living tibias, and of NPR2 phosphorylation in primary chondrocytes, showed that LB-100 counteracted FGF-induced dephosphorylation and inactivation of NPR2. In ex vivo experiments with Fgfr3Y367C/+ mice, the combination of BMN-111 and LB-100 increased bone length and cartilage area, restored chondrocyte terminal differentiation, and increased the proliferative growth plate area, more than BMN-111 alone. The combination treatment also reduced the abnormal elevation of MAP kinase activity in the growth plate of Fgfr3Y367C/+ mice and improved the skull base anomalies. Our results provide a proof of concept that a phosphatase inhibitor could be used together with an NPR2 agonist to enhance cGMP production as a therapy for ACH.

A disorder-related variant (E420K) of a PP2A-regulatory subunit (PPP2R5D) causes constitutively active AKT-mTOR signaling and uncoordinated cell growth.

Functional genomic approaches have facilitated the discovery of rare genetic disorders and improved efforts to decipher their underlying etiology. PPP2R5D-related disorder is an early childhood onset condition characterized by intellectual disability, hypotonia, autism-spectrum disorder, macrocephaly, and dysmorphic features. The disorder is caused by de novo single nucleotide changes in PPP2R5D, which generate heterozygous dominant missense variants. PPP2R5D is known to encode a B'-type (B'56δ) regulatory subunit of a PP2A-serine/threonine phosphatase. To help elucidate the molecular mechanisms altered in PPP2R5D-related disorder, we used a CRISPR-single-base editor to generate HEK-293 cells in which a single transition (c.1258G>A) was introduced into one allele, precisely recapitulating a clinically relevant E420K variant. Unbiased quantitative proteomic and phosphoproteomic analyses of endogenously expressed proteins revealed heterozygous-dominant changes in kinase/phosphatase signaling. These data combined with orthogonal validation studies revealed a previously unrecognized interaction of PPP2R5D with AKT in human cells, leading to constitutively active AKT-mTOR signaling, increased cell size, and uncoordinated cellular growth in E420K-variant cells. Rapamycin reduced cell size and dose-dependently reduced RPS6 phosphorylation in E420K-variant cells, suggesting that inhibition of mTOR1 can suppress both the observed RPS6 hyperphosphorylation and increased cell size. Together, our findings provide a deeper understanding of PPP2R5D and insight into how the E420K-variant alters signaling networks influenced by PPP2R5D. Our comprehensive approach, which combines precise genome editing, isobaric tandem mass tag labeling of peptides generated from endogenously expressed proteins, and concurrent liquid chromatography-mass spectrometry (LC-MS3), also provides a roadmap that can be used to rapidly explore the etiologies of additional genetic disorders.

S100A6 is a positive regulator of PPP5C-FKBP51-dependent regulation of endothelial calcium signaling.

ISOC is a cation current permeating the ISOC channel. In pulmonary endothelial cells, ISOC activation leads to formation of inter-endothelial cell gaps and barrier disruption. The immunophilin FK506-binding protein 51 (FKBP51), in conjunction with the serine/threonine protein phosphatase 5C (PPP5C), inhibits ISOC . Free PPP5C assumes an autoinhibitory state, which has low "basal" catalytic activity. Several S100 protein family members bind PPP5C increasing PPP5C catalytic activity in vitro. One of these family members, S100A6, exhibits a calcium-dependent translocation to the plasma membrane. The goal of this study was to determine whether S100A6 activates PPP5C in pulmonary endothelial cells and contributes to ISOC inhibition by the PPP5C-FKBP51 axis. We observed that S100A6 activates PPP5C to dephosphorylate tau T231. Following ISOC activation, cytosolic S100A6 translocates to the plasma membrane and interacts with the TRPC4 subunit of the ISOC channel. Global calcium entry and ISOC are decreased by S100A6 in a PPP5C-dependent manner and by FKBP51 in a S100A6-dependent manner. Further, calcium entry-induced endothelial barrier disruption is decreased by S100A6 dependent upon PPP5C, and by FKBP51 dependent upon S100A6. Overall, these data reveal that S100A6 plays a key role in the PPP5C-FKBP51 axis to inhibit ISOC and protect the endothelial barrier against calcium entry-induced disruption.

Development of a Synthetic 3-ketosteroid Δ1-dehydrogenase for the Generation of a Novel Catabolic Pathway Enabling Cholesterol Degradation in Human Cells.

Cholesterol is an essential component of membranes, which is acquired by cells via receptor-mediated endocytosis of lipoproteins or via de novo synthesis. In specialized cells, anabolic enzymes metabolize cholesterol, generating steroid hormones or bile acids. However, surplus cholesterol cannot be catabolized due to the lack of enzymes capable of degrading the cholestane ring. The inability to degrade cholesterol becomes evident in the development and progression of cardiovascular disease, where the accumulation of cholesterol/cholesteryl-esters in macrophages can elicit a maladaptive immune response leading to the development and progression of atherosclerosis. The discovery of cholesterol catabolic pathways in Actinomycetes led us to the hypothesis that if enzymes enabling cholesterol catabolism could be genetically engineered and introduced into human cells, the atherosclerotic process may be prevented or reversed. Comparison of bacterial enzymes that degrade cholesterol to obtain carbon and generate energy with the action of human enzymes revealed that humans lack a 3-ketosteroid Δ1-dehydrogenase (Δ1-KstD), which catalyzes the C-1 and C-2 desaturation of ring A. Here we describe the construction, heterologous expression, and actions of a synthetic humanized Δ1-KstD expressed in Hep3B and U-937 cells, providing proof that one of three key enzymes required for cholesterol ring opening can be functionally expressed in human cells.

The Antitumor Drug LB-100 Is a Catalytic Inhibitor of Protein Phosphatase 2A (PPP2CA) and 5 (PPP5C) Coordinating with the Active-Site Catalytic Metals in PPP5C.

LB-100 is an experimental cancer therapeutic with cytotoxic activity against cancer cells in culture and antitumor activity in animals. The first phase I trial (NCT01837667) evaluating LB-100 recently concluded that safety and efficacy parameters are favorable for further clinical testing. Although LB-100 is widely reported as a specific inhibitor of serine/threonine phosphatase 2A (PP2AC/PPP2CA:PPP2CB), we could find no experimental evidence in the published literature demonstrating the specific engagement of LB-100 with PP2A in vitro, in cultured cells, or in animals. Rather, the premise for LB-100 targeting PP2AC is derived from studies that measure phosphate released from a phosphopeptide (K-R-pT-I-R-R) or inferred from the ability of LB-100 to mimic activity previously reported to result from the inhibition of PP2AC by other means. PP2AC and PPP5C share a common catalytic mechanism. Here, we demonstrate that the phosphopeptide used to ascribe LB-100 specificity for PP2A is also a substrate for PPP5C. Inhibition assays using purified enzymes demonstrate that LB-100 is a catalytic inhibitor of both PP2AC and PPP5C. The structure of PPP5C cocrystallized with LB-100 was solved to a resolution of 1.65Å, revealing that the 7-oxabicyclo[2.2.1]heptane-2,3-dicarbonyl moiety coordinates with the metal ions and key residues that are conserved in both PP2AC and PPP5C. Cell-based studies revealed some known actions of LB-100 are mimicked by the genetic disruption of PPP5C These data demonstrate that LB-100 is a catalytic inhibitor of both PP2AC and PPP5C and suggest that the observed antitumor activity might be due to an additive effect achieved by suppressing both PP2A and PPP5C.

Inhibitors of Serine/Threonine Protein Phosphatases: Biochemical and Structural Studies Provide Insight for Further Development.

BACKGROUND: The reversible phosphorylation of proteins regulates many key functions in eukaryotic cells. Phosphorylation is catalyzed by protein kinases, with the majority of phosphorylation occurring on side chains of serine and threonine residues. The phosphomonoesters generated by protein kinases are hydrolyzed by protein phosphatases. In the absence of a phosphatase, the half-time for the hydrolysis of alkyl phosphate dianions at 25º C is over 1 trillion years; knon ~2 x 10-20 sec-1. Therefore, ser/thr phosphatases are critical for processes controlled by reversible phosphorylation. METHODS: This review is based on the literature searched in available databases. We compare the catalytic mechanism of PPP-family phosphatases (PPPases) and the interactions of inhibitors that target these enzymes. RESULTS: PPPases are metal-dependent hydrolases that enhance the rate of hydrolysis ([kcat/kM]/knon ) by a factor of ~1021, placing them among the most powerful known catalysts on earth. Biochemical and structural studies indicate that the remarkable catalytic proficiencies of PPPases are achieved by 10 conserved amino acids, DXH(X)~26DXXDR(X)~20- 26NH(X)~50H(X)~25-45R(X)~30-40H. Six act as metal-coordinating residues. Four position and orient the substrate phosphate. Together, two metal ions and the 10 catalytic residues position the phosphoryl group and an activated bridging water/hydroxide nucleophile for an inline attack upon the substrate phosphorous atom. The PPPases are conserved among species, and many structurally diverse natural toxins co-evolved to target these enzymes. CONCLUSION: Although the catalytic site is conserved, opportunities for the development of selective inhibitors of this important group of metalloenzymes exist.

Serine/threonine phosphatase 5 (PP5C/PPP5C) regulates the ISOC channel through a PP5C-FKBP51 axis.

Pulmonary endothelial cells express a store-operated calcium entry current ( Isoc), which contributes to inter-endothelial cell gap formation. Isoc is regulated by a heterocomplex of proteins that includes the immunophilin FKBP51. FKBP51 inhibits Isoc by mechanisms that are not fully understood. In pulmonary artery endothelial cells (PAECs) we have shown that FKBP51 increases microtubule polymerization, an event that is critical for Isoc inhibition by FKBP51. In neurons, FKBP51 promotes microtubule stability through facilitation of tau dephosphorylation. However, FKBP51 does not possess phosphatase activity. Protein phosphatase 5 (PP5C/PPP5C) can dephosphorylate tau, and similar to FKBP51, PP5C possesses tetratricopeptide repeats (TPR) that mediate interaction with heat shock protein-90 (HSP90) chaperone/scaffolding complexes. We therefore tested whether PP5C contributes to FKBP51-mediated inhibition of Isoc. Both siRNA-mediated suppression of PP5C expression in PAECs and genetic disruption of PP5C in HEK293 cells attenuate FKBP51-mediated inhibition of Isoc. Reintroduction of catalytically competent, but not catalytically inactive PP5C, restored FKBP51-mediated inhibition of Isoc. PAEC cell fractionation studies identified both PP5C and the ISOC heterocomplex in the same membrane fractions. Further, PP5C co-precipitates with TRPC4, an essential subunit of ISOC channel. Finally, to determine if PP5C is required for FKBP51-mediated inhibition of calcium entry-induced inter-endothelial cell gap formation, we measured gap area by wide-field microscopy and performed biotin gap quantification assay and electric cell-substrate impedance sensing (ECIS®). Collectively, the data presented indicate that suppression of PP5C expression negates the protective effect of FKBP51. These observations identify PP5C as a novel member of the ISOC heterocomplex that is required for FKBP51-mediated inhibition of Isoc.

PP1:Tautomycetin Complex Reveals a Path toward the Development of PP1-Specific Inhibitors.

Selective inhibitors for each serine/threonine phosphatase (PPP) are essential to investigate the biological actions of PPPs and to guide drug development. Biologically diverse organisms (e.g., cyanobacteria, dinoflagellates, beetles) produce structurally distinct toxins that are catalytic inhibitors of PPPs. However, most toxins exhibit little selectivity, typically inhibiting multiple family members with similar potencies. Thus, the use of these toxins as chemical tools to study the relationship between individual PPPs and their biological substrates, and how disruptions in these relationships contributes to human disease, is severely limited. Here, we show that tautomycetin (TTN) is highly selective for a single PPP, protein phosphatase 1 (PP1/PPP1C). Our structure of the PP1:TTN complex reveals that PP1 selectivity is defined by a covalent bond between TTN and a PP1-specific cysteine residue, Cys127. Together, these data provide key molecular insights needed for the development of novel probes targeting single PPPs, especially PP1.

An Ultra-High-Throughput Screen for Catalytic Inhibitors of Serine/Threonine Protein Phosphatases Types 1 and 5 (PP1C and PP5C).

Although there has been substantial success in the development of specific inhibitors for protein kinases, little progress has been made in the identification of specific inhibitors for their protein phosphatase counterparts. Inhibitors of PP1 and PP5 are desired as probes for research and to test their potential for drug development. We developed and miniaturized (1536-well plate format) nearly identical homogeneous, fluorescence intensity (FLINT) enzymatic assays to detect inhibitors of PP1 or PP5. The assays were used in an ultra-high-throughput screening (uHTS) campaign, testing >315,000 small-molecule compounds. Both assays demonstrated robust performance, with a Z' of 0.92 ± 0.03 and 0.95 ± 0.01 for the PP1 and PP5 assays, respectively. Screening the same library with both assays aided the identification of class inhibitors and assay artifacts. Confirmation screening and hit prioritization assays used [32P/33P]-radiolabel protein substrates, revealing excellent agreement between the FLINT and radiolabel assays. This screening campaign led to the discovery of four novel unrelated small-molecule inhibitors of PP1 and ~30 related small-molecule inhibitors of PP5. The results suggest that this uHTS approach is suitable for identifying selective chemical probes that inhibit PP1 or PP5 activity, and it is likely that similar assays can be developed for other PPP-family phosphatases.

Crystal structures and mutagenesis of PPP-family ser/thr protein phosphatases elucidate the selectivity of cantharidin and novel norcantharidin-based inhibitors of PP5C.

Cantharidin is a natural toxin and an active constituent in a traditional Chinese medicine used to treat tumors. Cantharidin acts as a semi-selective inhibitor of PPP-family ser/thr protein phosphatases. Despite sharing a common catalytic mechanism and marked structural similarity with PP1C, PP2AC and PP5C, human PP4C was found to be insensitive to the inhibitory activity of cantharidin. To explore the molecular basis for this selectivity, we synthesized and tested novel C5/C6-derivatives designed from quantum-based modeling of the interactions revealed in the co-crystal structures of PP5C in complex with cantharidin. Structure-activity relationship studies and analysis of high-resolution (1.25Å) PP5C-inhibitor co-crystal structures reveal close contacts between the inhibitor bridgehead oxygen and both a catalytic metal ion and a non-catalytic phenylalanine residue, the latter of which is substituted by tryptophan in PP4C. Quantum chemistry calculations predicted that steric clashes with the bulkier tryptophan side chain in PP4C would force all cantharidin-based inhibitors into an unfavorable binding mode, disrupting the strong coordination of active site metal ions observed in the PP5C co-crystal structures, thereby rendering PP4C insensitive to the inhibitors. This prediction was confirmed by inhibition studies employing native human PP4C. Mutation of PP5C (F446W) and PP1C (F257W), to mimic the PP4C active site, resulted in markedly suppressed sensitivity to cantharidin. These observations provide insight into the structural basis for the natural selectivity of cantharidin and provide an avenue for PP4C deselection. The novel crystal structures also provide insight into interactions that provide increased selectivity of the C5/C6 modifications for PP5C versus other PPP-family phosphatases.

Development and validation of a robust and sensitive assay for the discovery of selective inhibitors for serine/threonine protein phosphatases PP1α (PPP1C) and PP5 (PPP5C).

Protein phosphatase types 1 α (PP1α/PPP1C) and 5 (PP5/PPP5C) are members of the PPP family of serine/threonine protein phosphatases. PP1 and PP5 share a common catalytic mechanism, and several natural compounds, including okadaic acid, microcystin, and cantharidin, act as strong inhibitors of both enzymes. However, to date there have been no reports of compounds that can selectively inhibit PP1 or PP5, and specific or highly selective inhibitors for either PP1 or PP5 are greatly desired by both the research and pharmaceutical communities. Here we describe the development and optimization of a sensitive and robust (representative PP5C assay data: Z'=0.93; representative PP1Cα assay data: Z'=0.90) fluorescent phosphatase assay that can be used to simultaneously screen chemical libraries and natural product extracts for the presence of catalytic inhibitors of PP1 and PP5.

Protein phosphatases in pancreatic islets.

The prevalence of diabetes is increasing rapidly worldwide. A cardinal feature of most forms of diabetes is the lack of insulin-producing capability, due to the loss of insulin-producing ß-cells, impaired glucose-sensitive insulin secretion from the ß-cell, or a combination thereof, the reasons for which largely remain elusive. Reversible phosphorylation is an important and versatile mechanism for regulating the biological activity of many intracellular proteins, which, in turn, controls a variety of cellular functions. For instance, significant changes in protein kinase activities and in protein phosphorylation patterns occur subsequent to the stimulation of insulin release by glucose. Therefore, the molecular mechanisms regulating the phosphorylation of proteins involved in the insulin secretory process by the ß-cell have been extensively investigated. However, far less is known about the role and regulation of protein dephosphorylation by various protein phosphatases. Herein, we review extant data implicating serine/threonine and tyrosine phosphatases in various aspects of healthy and diabetic islet biology, ranging from control of hormonal stimulus-secretion coupling to mitogenesis and apoptosis.

Small G proteins Rac1 and Ras regulate serine/threonine protein phosphatase 5 (PP5)·extracellular signal-regulated kinase (ERK) complexes involved in the feedback regulation of Raf1.

Serine/threonine protein phosphatase 5 (PP5, PPP5C) is known to interact with the chaperonin heat shock protein 90 (HSP90) and is involved in the regulation of multiple cellular signaling cascades that control diverse cellular processes, such as cell growth, differentiation, proliferation, motility, and apoptosis. Here, we identify PP5 in stable complexes with extracellular signal-regulated kinases (ERKs). Studies using mutant proteins reveal that the formation of PP5·ERK1 and PP5·ERK2 complexes partially depends on HSP90 binding to PP5 but does not require PP5 or ERK1/2 activity. However, PP5 and ERK activity regulates the phosphorylation state of Raf1 kinase, an upstream activator of ERK signaling. Whereas expression of constitutively active Rac1 promotes the assembly of PP5·ERK1/2 complexes, acute activation of ERK1/2 fails to influence the phosphatase-kinase interaction. Introduction of oncogenic HRas (HRas(V12)) has no effect on PP5-ERK1 binding but selectively decreases the interaction of PP5 with ERK2, in a manner that is independent of PP5 and MAPK/ERK kinase (MEK) activity, yet paradoxically requires ERK2 activity. Additional studies conducted with oncogenic variants of KRas4B reveal that KRas(L61), but not KRas(V12), also decreases the PP5-ERK2 interaction. The expression of wild type HRas or KRas proteins fails to reduce PP5-ERK2 binding, indicating that the effect is specific to HRas(V12) and KRas(L61) gain-of-function mutations. These findings reveal a novel, differential responsiveness of PP5-ERK1 and PP5-ERK2 interactions to select oncogenic Ras variants and also support a role for PP5·ERK complexes in regulating the feedback phosphorylation of PP5-associated Raf1.

Mitogen-activated protein kinases and protein phosphatase 5 mediate glucocorticoid-induced cytotoxicity in pancreatic islets and ß-cells.

Glucocorticoid excess is associated with glucose intolerance and diabetes. In addition to inducing insulin resistance, glucocorticoids impair ß-cell function and cause ß-cell apoptosis. In this study we show that dexamethasone activates mitogen-activated protein kinases (MAPKs) signaling in MIN6 ß-cells, as evident by enhanced phosphorylation of p38 MAPK and c-Jun N-terminal kinase (JNK). In contrast, the integrated stress response pathway was inhibited by dexamethasone. A p38 MAPK inhibitor attenuated dexamethasone-induced apoptosis in ß-cells and isolated islets and decreased glucocorticoid receptor phosphorylation at S220. In contrast, a JNK inhibitor augmented DNA fragmentation and dexamethasone-induced formation of cleaved caspase 3. We also show that inhibition of protein phosphatase 5 (PP5) augments apoptosis in dexamethasone-exposed islets and ß-cells, with a concomitant activation of p38 MAPK. In conclusion, our data provide evidence that in islets and ß-cells, p38 MAPK and JNK phosphorylation work in concert with PP5 to regulate the cytotoxic effects exerted by glucocorticoids.