Steve Arkinstall

Dual specificity phosphatases: a gene family for control of MAP kinase function

Mitogen‐activated protein (MAP) kinases are important players in signal transduction pathways activated by a range of stimuli and mediate a number of physiological and pathological changes in cell function. MAP kinase activation requires phosphorylation on a threonine and tyrosine residue located within the activation loop of kinase subdomain VIII. This process is reversible even in the continued presence of activating stimuli, indicating that protein phosphatases provide an important mechanism for MAP kinase control. Dual specificity phosphatases (DSPs) are an emerging subclass of the protein tyrosine phosphatase (PTP) gene superfam‐ily, which appears to be selective for dephosphory‐lating the critical phosphothreonine and phosphoty‐rosine residues within MAP kinases. Some DSPs are localized to different subcellular compartments and moreover, certain family members appear highly selective for inactivating distinct MAP kinase iso‐forms. This enzymatic specificity is due in part to powerful catalytic activation of the DSP phosphatase after tight binding of its amino‐terminal to the target MAP kinase. DSP gene expression is induced strongly by various growth factors and/or cellular stresses, providing a sophisticated transcriptional mechanism for targeted inactivation of selected MAP kinase activities.—Camps, M., Nichols, A., Arkinstall, S. Dual specificity phosphatases: a gene family for control of MAP kinase function. FASEB J. 14, 6–16(1999)

Bcl-2 Undergoes Phosphorylation by c-Jun N-terminal Kinase/Stress-activated Protein Kinases in the Presence of the Constitutively Active GTP-binding Protein Rac1

We have studied the phosphorylation of the Bcl-2 family of proteins by different mitogen-activated protein (MAP) kinases. Purified Bcl-2 was found to be phosphorylated by the c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) p54-SAPKβ, and this is specific insofar as the extracellular signal-regulated kinase 1 (ERK1) and p38/RK/CSBP (p38) catalyzed only weak modification. Bcl-2 undergoes similar phosphorylation in COS-7 when coexpressed together with p54-SAPKβ and the constitutive Rac1 mutant G12V. This is seen by both 32PO4labeling and the appearance of five discrete Bcl-2 bands with reduced gel mobility. As anticipated, both intracellular p54-SAPKβ activation and Bcl-2 phosphorylation are blocked by co-transfection with the MAP kinase specific phosphatase MKP3/PYST1. MAP kinase specificity is also seen in COS-7 cells as Bcl-2 undergoes only weak phosphorylation when co-expressed with enzymatically activated ERK1 or p38. Four critical residues undergoing phosphorylation in COS-7 cells were identified by expression of the quadruple Bcl-2 point mutant T56A,S70A,T74A,S87A. Sequencing phosphopeptides derived from tryptic digests of Bcl-2 indicates that purified GST-p54-SAPKβ phosphorylates identical sitesin vitro. This is the first report of Bcl-2 phosphorylation by the JNK/SAPK class of MAP kinases and could indicate a key modification allowing control of Bcl-2 function by cell surface receptors, Rho family GTPases, and/or cellular stresses.

The Dual Specificity Phosphatases M3/6 and MKP-3 Are Highly Selective for Inactivation of Distinct Mitogen-activated Protein Kinases

The mitogen-activated protein (MAP) kinase family includes extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK) and p38/RK/CSBP (p38) as structurally and functionally distinct enzyme classes. Here we describe two new dual specificity phosphatases of the CL100/MKP-1 family that are selective for inactivating ERK or JNK/SAPK and p38 MAP kinases when expressed in COS-7 cells. M3/6 is the first phosphatase of this family to display highly specific inactivation of JNK/SAPK and p38 MAP kinases. Although stress-induced activation of p54 SAPKβ, p46 SAPKγ (JNK1) or p38 MAP kinases is abolished upon co-transfection with increasing amounts of M3/6 plasmid, epidermal growth factor-stimulated ERK1 is remarkably insensitive even to the highest levels of M3/6 expression obtained. In contrast to M3/6, the dual specificity phosphatase MKP-3 is selective for inactivation of ERK family MAP kinases. Low level expression of MKP-3 blocks totally epidermal growth factor-stimulated ERK1, whereas stress-induced activation of p54 SAPKβ and p38 MAP kinases is inhibited only partially under identical conditions. Selective regulation by M3/6 and MKP-3 was also observed upon chronic MAP kinase activation by constitutive p21ras GTPases. Hence, although M3/6 expression effectively blocked p54 SAPKβ activation by p21rac (G12V), ERK1 activated by p21ras (G12V) was insensitive to this phosphatase. ERK1 activation by oncogenic p21ras was, however, blocked totally by co-expression of MKP-3. This is the first report demonstrating reciprocally selective inhibition of different MAP kinases by two distinct dual specificity phosphatases.

Molecular cloning and functional characterization of a novel mitogen-activated protein kinase phosphatase, MKP-4

Extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK), and p38/RK/CSBP (p38) mitogen-activated protein (MAP) kinases are target enzymes activated by a wide range of cell-surface stimuli. Recently, a distinct class of dual specificity phosphatase has been shown to reverse activation of MAP kinases by dephosphorylating critical tyrosine and threonine residues. By searching the expressed sequence tag data base (dbEST) for homologues of known dual specificity phosphatases, we identified a novel partial human sequence for which we isolated a full-length cDNA (termed MKP-4). The deduced amino acid sequence of MKP-4 is most similar to MKP-X/PYST2 (61% identity) and MKP-3/PYST1 (57% identity), includes two N-terminal CH2 domains homologous to the cell cycle regulator Cdc25 phosphatase, and contains the extended active site sequence motif VXVHCXAGXSRSXTX3AYLM (where X is any amino acid) conserved in dual specificity phosphatases. MKP-4 produced in Escherichia coli catalyzes vanadate-sensitive breakdown of p-nitrophenyl phosphate as well as in vitro inactivation of purified ERK2. When expressed in COS-7 cells, MKP-4 blocks activation of MAP kinases with the selectivity ERK > p38 = JNK/SAPK. This cellular specificity is similar to MKP-3/PYST1, although distinct from hVH-5/M3-6 (JNK/SAPK = p38 >>> ERK). Northern analysis reveals a highly restricted tissue distribution with a single MKP-4 mRNA species of approximately 2.5 kilobases detected only in placenta, kidney, and embryonic liver. Immunocytochemical analysis showed MKP-4 to be present within cytosol although punctate nuclear staining co-localizing with promyelocytic protein was also observed in a subpopulation (10-20%) of cells. Chromosomal localization by analysis of DNAs from human/rodent somatic cell hybrids and a panel of radiation hybrids assign the human gene for MKP-4 to Xq28. The identification and characterization of MKP-4 highlights the emergence of an expanding family of structurally homologous dual specificity phosphatases possessing distinct MAP kinase specificity and subcellular localization as well as diverse patterns of tissue expression.

MKP5, a new member of the MAP kinase phosphatase family, which selectively dephosphorylates stress-activated kinases

Dual-specificity protein tyrosine phosphatases are a burgeoning family of enzymes, some of which, the MKPs, are implicated in the regulation of mitogen-activated protein (MAP) kinases. MKPs have been shown to reverse the activation of the MAP kinases by hydrolyzing phosphothreonine and phosphotyrosine residues present in the substrates. Here we describe the characterization of a novel member of the MKP family, MKP5. The MKP5 gene, which maps to human chromosome 1q32, is expressed tissue-specifically as two transcripts of approximately 3.4 and 2.4 kb in human liver and skeletal muscle. When expressed in mammalian cells, MKP5 blocks the enzymatic activation of MAP kinases with the selectivity p38≈JNK/SAPK>>ERK. Immunoprecipitation of endogenous MAP kinases by the catalytically inactive transfected MKP5 demonstrates that it preferentially binds to the p38 and JNK/SAPK kinases. These findings suggest that the selectivity of this phosphatase may be determined at least in part at the level of substrate binding.

Substrate Recognition Domains within Extracellular Signal-regulated Kinase Mediate Binding and Catalytic Activation of Mitogen-activated Protein Kinase Phosphatase-3

Mitogen-activated protein (MAP) kinase phosphatase-3 (MKP-3) is a dual specificity phosphatase that inactivates extracellular signal-regulated kinase (ERK) MAP kinases. This reflects tight and specific binding between ERK and the MKP-3 amino terminus with consequent phosphatase activation and dephosphorylation of the bound MAP kinase. We have used a series of p38/ERK chimeric molecules to identify domains within ERK necessary for binding and catalytic activation of MKP-3. These studies demonstrate that ERK kinase subdomains V-XI are necessary and sufficient for binding and catalytic activation of MKP-3. These domains constitute the major COOH-terminal structural lobe of ERK. p38/ERK chimeras possessing these regions display increased sensitivity to inactivation by MKP-3. These data also reveal an overlap between ERK domains interacting with MKP-3 and those known to confer substrate specificity on the ERK MAP kinase. Consistent with this, we show that peptides representing docking sites within the target substrates Elk-1 and p90 rsk inhibit ERK-dependent activation of MKP-3. In addition, abolition of ERK-dependent phosphatase activation following mutation of a putative kinase interactionmotif (KIM) within the MKP-3 NH2 terminus suggests that key sites of contact for the ERK COOH-terminal structural lobe include residues localized between the Cdc25 homology domains (CH2) found conserved between members of the DSP gene family.

Induction of the mitogen-activated protein kinase phosphatase MKP3 by nerve growth factor in differentiating PC12

In PC12 sympathetic neurons activation and nuclear translocation of ERK family MAP kinases plays an essential role in processes underlying nerve growth factor (NGF)‐dependent differentiation. We have recently cloned MKP‐3 as a novel dual specificity phosphatase displaying selectivity towards inactivation of the ERK1 and ERK2 MAP kinases. Here we report that in PC12 cells, MKP‐3 undergoes powerful and specific up‐regulation by NGF while a number of mitogens and cellular stresses are ineffective. NGF‐stimulated MKP‐3 expression appears after 1 h, is maximal at 3 h, and is sustained for 5 days. This coincides with a critical period of neurite outgrowth and terminal differentiation. Consistent with a role mediating inhibition of PC12 cell MAP kinases, NGF‐stimulated ERK2 activation was suppressed considerably following pretreatment with fibroblast growth factor and 9‐cis‐retinal, two additional differentiation factors found to induce powerfully MKP‐3 expression. Given the clear cytosolic localization of MKP3 in PC12 cells and sympathetic neurons, these results suggest a critical role for inactivating ERK MAP kinases in non‐nuclear compartments during essential stages of NGF‐mediated PC12 differentiation.

Palmitoylation but not the extreme amino-terminus of Gqα is required for coupling to the NK2 receptor

Gqα and G11α differ from other G protein α subunits in that they have unique, conserved 6 residue amino‐terminal extensions. Wild‐type and amino‐terminal mutants of Gqα expressed in COS cells were analyzed for their ability to functionally couple with co‐expressed neurokinin NK2 receptor. Wild‐type, T2A and Δ2–7 Gqα were able to stimulate agonist driven phospholipase C (PLC) activity in identical manners. Other activities of these two amino‐terminal mutants including aluminum fluoride stimulated PLC activity, palmitoylation, interaction with Gβγ subunits and GTPγS‐induced trypsin resistance are also similar to the wild‐type α subunit. This demonstrates that the NK2 receptor is able to functionally interact with the α subunit of Gq and that the first seven amino‐acids of Gqα are not required for any of the α subunit functions tested. In contrast to the T2A and Δ2–7 mutants, a C9,10A Gqα mutant was not able to couple to either the NK2 receptor or PLC, as assessed by high‐affinity agonist binding and activation of PLC either in intact cells or in vitro. The C9,10A protein was able to assume a GTPγS‐induced trypsin‐resistant conformation and partitioned primarily to the pelletable fraction in a manner similar to the wild‐type protein. However, it was not labeled with [3H]palmitic acid. This suggests that blocking palmitoylation at the amino‐terminus of Gqα results in a loss of functional activity which reflects an inability to interact with both the receptor and downstream signaling targets.

Regulated expression of dual specificity protein phosphatases in rat brain

ACTIVATED mitogen-activated protein (MAP) kinases play an essential role controlling many neuronal functions. Dual specificity protein phosphatases (DS-PTPs) elicit selective inactivation of MAP kinases and are under tight transcriptional control. We have studied expression of four DS-PTPs (MKP-1, MKP-X, MKP-3 and B23) in rat brain and examined changes during post-natal development and following kainic acid induced seizure activity. In normal adult brain these DS-PTPs exhibit a strikingly different expression pattern. Only MKP-1 was regulated during development with levels increased transiently (P15-P21) within the thalamus and somatosensory cortex. Following kainate treatment, MKP-1, MKP-3 and B23 all exhibit striking changes in expression within hippocampal subfields CA1-3 and dentate gyrus. Regulated transcription of DS-PTPs may play a critical role controlling MAP kinase dependent processes including synaptic remodeling and neuronal death.

c-Jun N-terminal kinase-3 (JNK3)/stress-activated protein kinase-β (SAPKβ) binds and phosphorylates the neuronal microtubule regulator SCG10

The neuronal growth‐associated protein SCG10 is enriched in the growth cones of neurons where it destabilizes microtubules and thus contributes to the dynamic assembly and disassembly of microtubules. Since its microtubule‐destabilizing activity is regulated by phosphorylation, SCG10 may link extracellular signals to rearrangements of the neuronal cytoskeleton. To identify signal transduction pathways that may lead to SCG10 phosphorylation, we tested a series of serine–threonine‐directed protein kinases that phosphorylate SCG10 in vitro. We demonstrate that purified SCG10 can be phosphorylated by two subclasses of mitogen‐activated protein (MAP) kinases, c‐Jun N‐terminal/stress‐activated protein kinase (JNK/SAPK) and p38 MAP kinase. Moreover, SCG10 was found to bind tightly and specifically to JNK3/SAPKβ. JNK3/SAPKβ phosphorylation occurs at Ser‐62 and Ser‐73, residues that result in reduced microtubule‐destabilizing activity for SCG10. Endogenous SCG10 also undergoes increased phosphorylation in sympathetic neurons at times of JNK3/SAPKβ activation following deprivation from nerve growth factor. Together these observations indicate that activation of JNK/SAPKs provides a pathway for phosphorylation of SCG10 and control of growth cone microtubule formation following neuronal exposure to cellular stresses.