A novel regulatory mechanism of MAP kinases activation and nuclear translocation mediated by PKA and the PTP-SL tyrosine phosphatase

Protein tyrosine phosphatase PTP-SL retains mitogen-activated protein (MAP) kinases in the cytoplasm in an inactive form by association through a kinase interaction motif (KIM) and tyrosine dephosphorylation. The related tyrosine phosphatases PTP-SL and STEP were phosphorylated by the cAMP-dependent protein kinase A (PKA). The PKA phosphorylation site on PTP-SL was identified as the Ser(231) residue, located within the KIM. Upon phosphorylation of Ser(231), PTP-SL binding and tyrosine dephosphorylation of the MAP kinases extracellular signal-regulated kinase (ERK)1/2 and p38alpha were impaired. Furthermore, treatment of COS-7 cells with PKA activators, or overexpression of the Calpha catalytic subunit of PKA, inhibited the cytoplasmic retention of ERK2 and p38alpha by wild-type PTP-SL, but not by a PTP-SL S231A mutant. These findings support the existence of a novel mechanism by which PKA may regulate the activation and translocation to the nucleus of MAP kinases.     

Interaction of mitogen-activated protein kinases with the kinase interaction motif of the tyrosine phosphatase PTP-SL provides substrate specificity and retains ERK2 in the cytoplasm.

ERK1 and ERK2 associate with the tyrosine phosphatase PTP-SL through a kinase interaction motif (KIM) located in the juxtamembrane region of PTP-SL. A glutathione S-transferase (GST)-PTP-SL fusion protein containing the KIM associated with ERK1 and ERK2 as well as with p38/HOG, but not with the related JNK1 kinase or with protein kinase A or C. Accordingly, ERK2 showed in vitro substrate specificity to phosphorylate GST-PTP-SL in comparison with GST-c-Jun. Furthermore, tyrosine dephosphorylation of ERK2 by the PTP-SLDeltaKIM mutant was impaired. The in vitro association of ERK1/2 with GST-PTP-SL was highly stable; however, low concentrations of nucleotides partially dissociated the ERK1/2.PTP-SL complex. Partial deletions of the KIM abrogated the association of PTP-SL with ERK1/2, indicating that KIM integrity is required for interaction. Amino acid substitution analysis revealed that Arg and Leu residues within the KIM are essential for the interaction and suggested a regulatory role for Ser(231). Finally, coexpression of PTP-SL and ERK2 in COS-7 cells resulted in the retention of ERK2 in the cytoplasm in a KIM-dependent manner. Our results demonstrate that the noncatalytic region of PTP-SL associates with mitogen-activated protein kinases with high affinity and specificity, providing a mechanism for substrate specificity, and suggest a role for PTP-SL in the regulation of mitogen-activated protein kinase translocation to the nucleus upon activation.     

Crystal structure of PTP-SL/PTPBR7 catalytic domain: implications for MAP kinase regulation

Protein tyrosine phosphatases PTP-SL and PTPBR7 are isoforms belonging to cytosolic membrane-associated and to receptor-like PTPs (RPTPs), respectively. They represent a new family of PTPs with a major role in activation and translocation of MAP kinases. Specifically, the complex formation between PTP-SL and ERK2 involves an unusual interaction leading to the phosphorylation of PTP-SL by ERK2 at Thr253 and the inactivating dephosphorylation of ERK2 by PTP-SL. This interaction is strictly dependent upon a kinase interaction motif (KIM) (residues 224-239) situated at the N terminus of the PTP-SL catalytic domain. We report the first crystal structure of the catalytic domain for a member of this family (PTP-SL, residues 254-549, identical with residues 361-656 of PTPBR7), providing an example of an RPTP with single cytoplasmic domain, which is monomeric, having an unhindered catalytic site. In addition to the characteristic PTP-core structure, PTP-SL has an N-terminal helix, possibly orienting the KIM motif upon interaction with the target ERK2. An unusual residue in the catalytically important WPD loop promotes formation of a hydrophobically and electrostatically stabilised clamp. This could induce increased rigidity to the WPD loop and therefore reduced catalytic activity, in agreement with our kinetic measurements. A docking model based on the PTP-SL structure suggests that, in the complex with ERK2, the phosphorylation of PTP-SL should be accomplished first. The subsequent dephosphorylation of ERK2 seems to be possible only if a conformational rearrangement of the two interacting partners takes place.     

PTP-SL and STEP protein tyrosine phosphatases regulate the activation of the extracellular signal-regulated kinases ERK1 and ERK2 by association through a kinase interaction motif.

Protein kinases and phosphatases regulate the activity of extracellular signal-regulated kinases 1 and 2 (ERK1/2) by controlling the phosphorylation of specific residues. We report the physical and functional association of ERK1/2 with the PTP-SL and STEP protein tyrosine phosphatases (PTPs). Upon binding, the N-terminal domains of PTP-SL and STEP were phosphorylated by ERK1/2, whereas these PTPs dephosphorylated the regulatory phosphotyrosine residues of ERK1/2 and inactivated them. A sequence of 16 amino acids in PTP-SL was identified as being critical for ERK1/2 binding and termed kinase interaction motif (KIM) (residues 224-239); it was shown to be required for phosphorylation of PTP-SL by ERK1/2 at Thr253. Co-expression of ERK2 with catalytically active PTP-SL in COS-7 cells impaired the EGF-induced activation of ERK2, whereas a PTP-SL mutant, lacking PTP activity, increased the ERK2 response to EGF. This effect was dependent on the presence of the KIM on PTP-SL. Furthermore, ERK1/2 activity was downregulated in 3T3 cells stably expressing PTP-SL. Our findings demonstrate the existence of a conserved ERK1/2 interaction motif within the cytosolic non-catalytic domains of PTP-SL and STEP, which is required for the regulation of ERK1/2 activity and for phosphorylation of the PTPs by these kinases. Our findings suggest that PTP-SL and STEP act as physiological regulators of the ERK1/2 signaling pathway.     

Molecular determinants of substrate recognition in hematopoietic protein-tyrosine phosphatase

The extracellular signal-regulated protein kinase 2 (ERK2) plays a central role in cellular proliferation and differentiation. Full activation of ERK2 requires dual phosphorylation of Thr183 and Tyr185 in the activation loop. Tyr185 dephosphorylation by the hematopoietic protein-tyrosine phosphatase (HePTP) represents an important mechanism for down-regulating ERK2 activity. The bisphosphorylated ERK2 is a highly efficient substrate for HePTP with a kcat/Km of 2.6 x 10(6) m(-1) s(-1). In contrast, the kcat/Km values for the HePTP-catalyzed hydrolysis of Tyr(P) peptides are 3 orders of magnitude lower. To gain insight into the molecular basis for HePTP substrate specificity, we analyzed the effects of altering structural features unique to HePTP on the HePTP-catalyzed hydrolysis of p-nitrophenyl phosphate, Tyr(P) peptides, and its physiological substrate ERK2. Our results suggest that substrate specificity is conferred upon HePTP by both negative and positive selections. To avoid nonspecific tyrosine dephosphorylation, HePTP employs Thr106 in the substrate recognition loop as a key negative determinant to restrain its protein-tyrosine phosphatase activity. The extremely high efficiency and fidelity of ERK2 dephosphorylation by HePTP is achieved by a bipartite protein-protein interaction mechanism, in which docking interactions between the kinase interaction motif in HePTP and the common docking site in ERK2 promote the HePTP-catalyzed ERK2 dephosphorylation (approximately 20-fold increase in kcat/Km) by increasing the local substrate concentration, and second site interactions between the HePTP catalytic site and the ERK2 substrate-binding region enhance catalysis (approximately 20-fold increase in kcat/Km) by organizing the catalytic residues with respect to Tyr(P)185 for optimal phosphoryl transfer.     

Visualizing active-site dynamics in single crystals of HePTP: opening of the WPD loop involves coordinated movement of the E loop

Phosphotyrosine hydrolysis by protein tyrosine phosphatases (PTPs) involves substrate binding by the PTP loop and closure over the active site by the WPD loop. The E loop, located immediately adjacent to the PTP and WPD loops, is conserved among human PTPs in both sequence and structure, yet the role of this loop in substrate binding and catalysis is comparatively unexplored. Hematopoietic PTP (HePTP) is a member of the kinase interaction motif (KIM) PTP family. Compared to other PTPs, KIM-PTPs have E loops that are unique in both sequence and structure. In order to understand the role of the E loop in the transition between the closed state and the open state of HePTP, we identified a novel crystal form of HePTP that allowed the closed-state-to-open-state transition to be observed within a single crystal form. These structures, which include the first structure of the HePTP open state, show that the WPD loop adopts an ‘atypically open’ conformation and, importantly, that ligands can be exchanged at the active site, which is critical for HePTP inhibitor development. These structures also show that tetrahedral oxyanions bind at a novel secondary site and function to coordinate the PTP, WPD, and E loops. Finally, using both structural and kinetic data, we reveal a novel role for E-loop residue Lys182 in enhancing HePTP catalytic activity through its interaction with Asp236 of the WPD loop, providing the first evidence for the coordinated dynamics of the WPD and E loops in the catalytic cycle, which, as we show, is relevant to multiple PTP families.     

FYVE-DSP1, a dual-specificity protein phosphatase containing an FYVE domain

Dual-specificity protein phosphatases (DSPs) dephosphorylate proteins at Ser/Thr and Tyr. FYVE domain is a double zinc finger motif which specifically binds phosphatidylinositol(3)-phosphate. Here, we report a novel dual specificity phosphatase that contains a FYVE domain at the C-terminus. We designate the protein FYVE-DSP1. Molecular cloning yielded three isoforms of the enzyme presumably derived from alternate RNA splicing. Sequence alignment revealed that the catalytic phosphatase domain of FYVE-DSP1 closely resembled that of myotubularin, while its FYVE domain has all the conserved amino acid residues found in other proteins of the same family. Recombinant FYVE-DSP1 is partitioned in both cytosolic and membrane fractions. It dephosphorylates proteins phosphorylated on Ser, Thr, and Tyr residues and low molecular weight phosphatase substrate para-nitrophenylphosphate. It shows typical characteristics of other DSPs and protein tyrosine phosphatases (PTPs). These include inhibition by sodium vanadate and pervanadate, pH dependency, and inactivation by mutation of the key cysteinyl residue at the phosphatase signature motif. Finally, PCR analyses demonstrated that FYVE-DSP1 is widely distributed in human tissues but different spliced forms expressed differently.     

Phosphotyrosine-specific phosphatase PTP-SL regulates the ERK5 signaling pathway

The duration and the magnitude of mitogen-activated protein kinase (MAPK) activation specifies signal identity and thus allows the regulation of diverse cellular functions by the same kinase cascade. A tight and finely tuned regulation of MAPK activity is therefore critical for the definition of a specific cellular response. We investigated the role of tyrosine-specific phosphatases (PTPs) in the regulation of ERK5. Although unique in its structure, ERK5 is activated in analogy to other MAPKs by dual phosphorylation of threonine and tyrosine residues in its activation motif. In this study we concentrated on whether and how PTP-SL, a kinase-interacting motif-containing PTP, might be involved in the down-regulation of the ERK5 signal. We found that both proteins interact directly with each other in vitro and in intact cells, resulting in mutual modulation of their enzymatic activities. PTP-SL is a substrate of ERK5 and independent of phosphorylation binding to the kinase enhances its catalytic phosphatase activity. On the other hand, interaction with PTP-SL not only down-regulates endogenous ERK5 activity but also effectively impedes the translocation of ERK5 to the nucleus. These findings indicate a direct regulatory influence of PTP-SL on the ERK5 pathway and corresponding downstream responses of the cell.     

Structural basis of p38α regulation by hematopoietic tyrosine phosphatase

MAP kinases regulate essential cellular events, including cell growth, differentiation and inflammation. The solution structure of a complete MAPK-MAPK-regulatory protein complex, p38α-HePTP, was determined, enabling a comprehensive investigation of the molecular basis of specificity and fidelity in MAPK regulation. Structure determination was achieved by combining NMR spectroscopy and small-angle X-ray scattering data with a new ensemble calculation-refinement procedure. We identified 25 residues outside of the HePTP kinase interaction motif necessary for p38α recognition. The complex adopts an extended conformation in solution and rarely samples the conformation necessary for kinase deactivation. Complex formation also does not affect the N-terminal lobe, the activation loop of p38α or the catalytic domain of HePTP. Together, these results show how the downstream tyrosine phosphatase HePTP regulates p38α and provide for fundamentally new insights into MAPK regulation and specificity.     

Crystal structure of the catalytic domain of human MAP kinase phosphatase 5: structural insight into constitutively active phosphatase

MAP kinase phosphatase 5 (MKP5) is a member of the mitogen-activated protein kinase phosphatase (MKP) family and selectively dephosphorylates JNK and p38. We have determined the crystal structure of the catalytic domain of human MKP5 (MKP5-C) to 1.6 A. In previously reported MKP-C structures, the residues that constitute the active site are seriously deviated from the active conformation of protein tyrosine phosphatases (PTPs), which are accompanied by low catalytic activity. High activities of MKPs are achieved by binding their cognate substrates, representing substrate-induced activation. However, the MKP5-C structure adopts an active conformation of PTP even in the absence of its substrate binding, which is consistent with the previous results that MKP5 solely possesses the intrinsic activity. Further, we identify a sequence motif common to the members of MKPs having low catalytic activity by comparing structures and sequences of other MKPs. Our structural information provides an explanation of constitutive activity of MKP5 as well as the structural insight into substrate-induced activation occurred in other MKPs.     

Evolution of the multifunctional protein tyrosine phosphatase family

The protein tyrosine phosphatase (PTP) family plays a central role in signal transduction pathways by controlling the phosphorylation state of serine, threonine, and tyrosine residues. PTPs can be divided into dual specificity phosphatases and the classical PTPs, which can comprise of one or two phosphatase domains. We studied amino acid substitutions at functional sites in the phosphatase domain and identified putative noncatalytic phosphatase domains in all subclasses of the PTP family. The presence of inactive phosphatase domains in all subclasses indicates that they were invented multiple times in evolution. Depending on the domain composition, loss of catalytic activity can result in different consequences for the function of the protein. Inactive single-domain phosphatases can still specifically bind substrate and protect it from dephosphorylation by other phosphatases. The inactive domains of tandem phosphatases can be further subdivided. The first class is more conserved, still able to bind phosphorylated tyrosine residues and might recruit multiphosphorylated substrates for the adjacent active domain. The second has accumulated several variable amino acid substitutions in the catalytic center, indicating a complete loss of tyrosine-binding capabilities. To study the impact of substitutions in the catalytic center to the evolution of the whole domain, we examined the evolutionary rates for each individual site and compared them between the classes. This analysis revealed a release of evolutionary constraint for multiple sites surrounding the catalytic center only in the second class, emphasizing its difference in function compared with the first class. Furthermore, we found a region of higher conservation common to both domain classes, suggesting a new regulatory center. We discuss the influence of evolutionary forces on the development of the phosphatase domain, which has led to additional functions, such as the specific protection of phosphorylated tyrosine residues, substrate recruitment, and regulation of the catalytic activity of adjacent domains.     

Crystal structure of the human dual specificity phosphatase 1 catalytic domain

The dual specificity phosphatase DUSP1 was the first mitogen activated protein kinase phosphatase (MKP) to be identified. It dephosphorylates conserved tyrosine and threonine residues in the activation loops of mitogen activated protein kinases ERK2, JNK1 and p38‐alpha. Here, we report the crystal structure of the human DUSP1 catalytic domain at 2.49 Å resolution. Uniquely, the protein was crystallized as an MBP fusion protein in complex with a monobody that binds to MBP. Sulfate ions occupy the phosphotyrosine and putative phosphothreonine binding sites in the DUSP1 catalytic domain.     

ERK2 shows a restrictive and locally selective mechanism of recognition by its tyrosine phosphatase inactivators not shared by its activator MEK1

The two regulatory residues that control the enzymatic activity of the mitogen-activated protein (MAP) kinase ERK2 are phosphorylated by the unique MAP kinase kinases MEK1/2 and dephosphorylated by several tyrosine-specific and dual specificity protein phosphatases. Selective docking interactions facilitate these phosphorylation and dephosphorylation events, controlling the specificity and duration of the MAP kinase activation-inactivation cycles. We have analyzed the contribution of specific residues of ERK2 in the physical and functional interaction with the ERK2 phosphatase inactivators PTP-SL and MKP-3 and with its activator MEK1. Single mutations in ERK2 that abrogated the dephosphorylation by endogenous tyrosine phosphatases from HEK293 cells still allowed efficient phosphorylation by endogenous MEK1/2. Discrete ERK2 mutations at the ERK2 docking groove differentially affected binding and inactivation by PTP-SL and MKP-3. Remarkably, the cytosolic retention of ERK2 by its activator MEK1 was not affected by any of the analyzed ERK2 single amino acid substitutions. A chimeric MEK1 protein, containing the kinase interaction motif of PTP-SL, bound tightly to ERK2 through its docking groove and behaved as a gain-of-function MAP kinase kinase that hyperactivated ERK2. Our results provide evidence that the ERK2 docking groove is more restrictive and selective for its tyrosine phosphatase inactivators than for MEK1/2 and indicate that distinct ERK2 residues modulate the docking interactions with activating and inactivating effectors.     

Structural and functional effects of disease-causing amino acid substitutions affecting residues Ala72 and Glu76 of the protein tyrosine phosphatase SHP-2

Mutations of the protein tyrosine phosphatase SHP-2 are implicated in human diseases, causing Noonan syndrome (NS) and related developmental disorders or contributing to leukemogenesis depending on the specific amino acid substitution involved. SHP-2 is composed by a catalytic (PTP) and two regulatory (N-SH2 and C-SH2) domains that bind to signaling partners and control the enzymatic activity by limiting the accessibility of the catalytic site. Wild type SHP-2 and four disease-associated mutants recurring in hematologic malignancies (Glu76Lys and Ala72Val) or causing NS (Glu76Asp and Ala72Ser), with affected residues located in the PTP-interacting region of the N-SH2 domain, were analyzed by molecular dynamics simulations and in vitro biochemical assays. Simulations demonstrate that mutations do not affect significantly the conformation of the N-SH2 domain. Rather they destabilize the interaction of this domain with the catalytic site, with more evident effects in the two leukemia associated mutants. Consistent with this structural evidence, mutants exhibit an increased level of basal phosphatase activity in the order Glu76Lys > Ala72Val > Glu76Asp > Ala72Ser > WT. The experimental data also show that the mutants with higher basal activity are more responsive to an activating phosphopeptide. A thermodynamic analysis demonstrates that an increase in the overall phosphopeptide affinity of mutants can be explained by a shift in the equilibrium between the inactive and active SHP-2 structure. These data support the view that an increase in the affinity of SHP-2 for its binding partners, caused by destabilization of the closed, inactive conformation, rather than protein basal activation per se, would represent the molecular mechanism, leading to pathogenesis in these mutants.     

Serine phosphorylation of protein tyrosine phosphatase (PTP1B) in HeLa cells in response to analogues of cAMP or diacylglycerol plus okadaic acid

The major intracellular protein tyrosine phosphatase (PTP1B) is a 50kDa protein, localized to the endoplasmic reticulum. This PTP is recovered in the particulate fraction of mamalian cells and can be solubilized as a complex of 150 kDa by extraction with non-ionic detergents. Previous work from this laboratory implicated phosphorylation of serine/threonine residues in the regulation of this PTP. Activity was several-fold higher in cells treated with activators of cAMP-dependent or Ca²⁺/phospholipid-dependent protein kinases or inhibitors of protein phosphatase 2A. Here we show that these treatments result in more than an 8-fold increase in the phosphorylation of the 50kDa PTP catalytic subunit within the 150kDa form of the phosphatase in HeLa cells. The phosphorylation occurred exclusively on serine residues, and the same tryptic and cyanogen bromide,³²P-phosphopeptides were recovered in the PTP from control and stimulated cells. Either multiple kinases phosphorylate a common site in the PTP1B, or a single kinase is activated downstream of cAMP- and Ca²⁺/phospholipid-dependent kinases. The results indicate that phosphorylation of a serine residue in the segment 283–364, probably serine 352 in the sequence Lys-Gly-Ser-Pro-Leu, occurs in response to cell stimulation. Phosphorylation in this region of PTP1B, between the N-terminal catalytic domain and the C-terminal membrane localization segment, is proposed to regulate phosphatase activity.     

Determinants of the tumor suppressor INPP4B protein and lipid phosphatase activities

The tumor suppressor INPP4B is an important regulator of phosphatidyl-inositol signaling in the cell. Reduced INPP4B expression is associated with poor outcomes for breast, prostate, and ovarian cancer patients. INPP4B contains a CX₅R catalytic motif characteristic of dual-specificity phosphatases, such as PTEN. Lipid phosphatase activity of INPP4B has previously been described. In this report we show that INPP4B can dephosphorylate para-nitrophenyl phosphate (pNPP) and 6,8-difluoro-4-methylumbelliferyl (DiFMUP), synthetic phosphotyrosine analogs, suggesting that INPP4B has protein tyrosine phosphatase (PTP) activity. Using mutagenesis, we examined the functional role of specific amino acids within the INPP4B C₈₄₂KSAKDR catalytic site. The K843M mutant displayed increased pNPP hydrolysis, the K846M mutant lost lipid phosphatase activity with no effect on PTP activity, and the D847E substitution ablated PTP activity and significantly reduced lipid phosphatase activity. Further, we show that INPP4B but not PTEN is able to reduce tyrosine phosphorylation of Akt1 and both the lipid and PTP activity of INPP4B likely contribute to the reduction of Akt1 phosphorylation. Taken together our data identified key residues in the INPP4B catalytic domain associated with lipid and protein phosphatase activities and found a robust downstream target regulated by INPP4B but not PTEN.     

Structural Basis for the Regulation of the Mitogen-activated Protein (MAP) Kinase p38α by the Dual Specificity Phosphatase 16 MAP Kinase Binding Domain in Solution

Mitogen-activated protein kinases (MAPKs) fulfill essential biological functions and are key pharmaceutical targets. Regulation of MAPKs is achieved via a plethora of regulatory proteins including activating MAPKKs and an abundance of deactivating phosphatases. Although all regulatory proteins use an identical interaction site on MAPKs, the common docking and hydrophobic pocket, they use distinct kinase interaction motif (KIM or D-motif) sequences that are present in linear, peptide-like, or well folded protein domains. It has been recently shown that a KIM-containing MAPK-specific dual specificity phosphatase DUSP10 uses a unique binding mode to interact with p38α. Here we describe the interaction of the MAPK binding domain of DUSP16 with p38α and show that despite belonging to the same dual specificity phosphatase (DUSP) family, its interaction mode differs from that of DUSP10. Indeed, the DUSP16 MAPK binding domain uses an additional helix, α-helix 4, to further engage p38α. This leads to an additional interaction surface on p38α. Together, these structural and energetic differences in p38α engagement highlight the fine-tuning necessary to achieve MAPK specificity and regulation among multiple regulatory proteins.     

Molecular mechanism of ERK dephosphorylation by striatal‐enriched protein tyrosine phosphatase

Striatal‐enriched tyrosine phosphatase (STEP) is an important regulator of neuronal synaptic plasticity, and its abnormal level or activity contributes to cognitive disorders. One crucial downstream effector and direct substrate of STEP is extracellular signal‐regulated protein kinase (ERK), which has important functions in spine stabilisation and action potential transmission. The inhibition of STEP activity toward phospho‐ERK has the potential to treat neuronal diseases, but the detailed mechanism underlying the dephosphorylation of phospho‐ERK by STEP is not known. Therefore, we examined STEP activity toward para‐nitrophenyl phosphate, phospho‐tyrosine‐containing peptides, and the full‐length phospho‐ERK protein using STEP mutants with different structural features. STEP was found to be a highly efficient ERK tyrosine phosphatase that required both its N‐terminal regulatory region and key residues in its active site. Specifically, both kinase interaction motif (KIM) and kinase‐specific sequence of STEP were required for ERK interaction. In addition to the N‐terminal kinase‐specific sequence region, S245, hydrophobic residues L249/L251, and basic residues R242/R243 located in the KIM region were important in controlling STEP activity toward phospho‐ERK. Further kinetic experiments revealed subtle structural differences between STEP and HePTP that affected the interactions of their KIMs with ERK. Moreover, STEP recognised specific positions of a phospho‐ERK peptide sequence through its active site, and the contact of STEP F311 with phospho‐ERK V205 and T207 were crucial interactions. Taken together, our results not only provide the information for interactions between ERK and STEP, but will also help in the development of specific strategies to target STEP‐ERK recognition, which could serve as a potential therapy for neurological disorders.