Abl-kinase-sensitive levels of ERK5 and its intrinsic basal activity contribute to leukaemia cell survival

It is well established that the mitogen-activated protein kinase (MAPK) signal is regulated through phosphorylation-dependent activation by the three-tiered MAPK cascade. However, our studies on the interaction of the MAPK ERK5 with the tyrosine kinase c-Abl and its oncogenic variants v-Abl and Bcr/Abl disclosed an alternative aspect of regulation. Independent of the MAPK cascade, Abl kinases were able to regulate the cellular amount of ERK5, at least in part, by stabilizing the protein. The resulting level of ERK5 and its intrinsic basal activity, but not necessarily its activation, were essential and sufficient to increase transformation by v-Abl and to mediate survival of Bcr/Abl-expressing leukaemia cells. These results suggest that the ability to regulate the cellular abundance of ERK5 contributes to the oncogenic potential of Abl kinases.     

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.     

Endogenous δ-Opioid and ORL1 Receptors Couple to Phosphorylation and Activation of p38 MAPK in NG108-15 Cells and This Is Regulated by Protein Kinase A and Protein Kinase C

The p38 mitogen-activated protein kinase (MAPK) cascade transduces multiple extracellular signals from cell surface to nucleus and is employed in cellular responses to cellular stresses and apoptotic regulation. The involvement of the p38 MAPK cascade in opioid- and opioid receptor-like receptor-1 (ORL1) receptor-mediated signal transduction was examined in NG108-15 neuroblastoma x glioma hybrid cells. Stimulation of endogenous delta-opioid receptor (DOR) or ORL1 resulted in activation of p38 MAPK. It also induced the activation of extracellular signal-regulated kinases (ERKs), another member of the MAPK family, with slower kinetics. Activation of p38 MAPK was abolished by selective antagonists of DOR or ORL1, pretreatment with pertussis toxin, or SB203580, a specific inhibitor of p38 MAPK. Inhibition of p38 MAPK had no significant effect on opioid-induced ERK activation, indicating that p38 MAPK activity was not required for ERK activation, though its stimulation preceded ERK activation. Inhibition of protein kinase A (PKA) strongly diminished p38 activation mediated by DOR or ORL1 but had no significant effect on ERK activation, and protein kinase C (PKC) inhibitors potentiated stimulation of p38 while inhibiting activation of ERKs. Taken together, our results provide the first evidence for coupling of DOR and ORL1 to the p38 MAPK cascade and clearly demonstrate that receptor-mediated activation of p38 MAPK both involves PKA and is negatively regulated by PKC.     

ERK7 is an autoactivated member of the MAPK family

Extracellular signal-regulated kinase 7 (ERK7) shares significant sequence homology with other members of the ERK family of signal transduction proteins, including the signature TEY activation motif. However, ERK7 has several distinguishing characteristics. Unlike other ERKs, ERK7 has been shown to have significant constitutive activity in serum-starved cells, which is not increased further by extracellular stimuli that typically activate other members of the mitogen-activated protein kinase (MAPK) family. On the other hand, ERK7's activation state and kinase activity appear to be regulated by its ability to utilize ATP and the presence of its extended C-terminal region. In this study, we investigated the mechanism of ERK7 activation. The results suggest that 1) MAPK kinase (MEK) inhibitors do not suppress ERK7 kinase activity; 2) intramolecular autophosphorylation is sufficient for activation of ERK7 in the absence of an upstream MEK; and 3) multiple regions of the C-terminal domain of ERK7 regulate its kinase activity. Taken together, these results indicate that autophosphorylation is sufficient for ERK7 activation and that the C-terminal domain regulates its kinase activity through multiple interactions.     

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.     

The ERK cascade

Sequential activation of protein kinases within the mitogen-activated protein kinase (MAPK) cascades is a common mechanism of signal transduction in many cellular processes. Four such cascades have been elucidated thus far, and named according to their MAPK tier component as the ERK1/2, JNK, p38MAPK, and ERK5 cascades. These cascades cooperate in transmitting various extracellular signals, and thus control cellular processes such as proliferation, differentiation, development, stress response, and apoptosis. Here we describe the classic ERK1/2 cascade, and concentrate mainly on the properties of MEK1/2 and ERK1/2, including their mode of regulation and their role in various cellular processes and in oncogenesis. This cascade may serve as a prototype of the other MAPK cascades, and the study of this cascade is likely to contribute to the understanding of mitogenic and other processes in many cell lines and tissues.     

蛋白激酶MSK在表观遗传学调控中的作用及其生物学意义

丝裂原和应激激活的蛋白激酶(MSK)是一类核内丝/苏氨酸蛋白激酶,参与丝裂原激活蛋白激酶(MAPK)信号通路介导的下游基因转录调控和表观遗传学调控.首先,MSK是MAPK通路的下游媒介分子.在丝裂原或应激刺激下,p38或ERK激酶通过级联磷酸化激活MSK蛋白.然后,活化的MSK介导转录因子磷酸化活化和组蛋白H3的10位丝氨酸磷酸化.MSK介导的组蛋白H3磷酸化,可引发组蛋白乙酰化和甲基化修饰的动态变化,相互协同或拮抗,开放染色质结构,利于诱导型基因的表达.除组蛋白H3外,MSK直接磷酸化的下游底物还包括CREB、NF-κB等转录因子以及多个非转录相关蛋白.因此,MSK能在多层次调控基因表达和细胞功能,广泛参与肿瘤转化、炎症反应、神经突触可塑性以及心肌肥大等生物学事件.本文将简要介绍MSK蛋白的研究进展,探讨其在转录调控、表观遗传学修饰等生物学事件中的作用.     

Inactivation of mitogen-activated protein kinases by a mammalian tyrosine-specific phosphatase, PTPBR7

Mitogen-activated protein kinase (MAPK) is inactivated through dephosphorylation of tyrosyl and threonyl regulatory sites. In yeast, both dual-specificity and tyrosine-specific phosphatases are involved in dephosphorylation. In mammals, however, no tyrosine-specific phosphatase has been identified molecularly to dephosphorylate MAPK in vivo. Recently, we and others have cloned a murine tyrosine-specific phosphatase, PTPBR7/PTP-SL, which is expressed predominantly in the brain. Here we report inactivation of the extracellular signal-regulated kinase (ERK) family MAPK by PTPBR7. PTPBR7 made complexes with ERK1/ERK2 in vivo and dephosphorylated ERK1 in vitro. When overexpressed in mammalian cells, wild-type PTPBR7 suppressed the phosphorylation and activation of ERK by epidermal growth factor (EGF), nerve growth factor (NGF), and constitutively active MEK1, a mutant MAPK kinase. In contrast, catalytically inactive and ERK-binding-deficient mutants revealed little inhibition on the ERK cascade. These results indicate that PTPBR7 suppresses MAPK directly in vivo.     

Role of a mitogen-activated protein kinase pathway in the induction of phase II detoxifying enzymes by chemicals.

Mitogen-activated protein kinase (MAPK) cascades are activated by diverse extracellular signals and participate in the regulation of an array of cellular programs. In this study, we investigated the roles of MAPKs in the induction of phase II detoxifying enzymes by chemicals. Treatment of human hepatoma (HepG2) and murine hepatoma (Hepa1c1c7) cells with tert-butylhydroquinone (tBHQ) or sulforaphane (SUL), two potent phase II enzyme inducers, stimulated the activity of extracellular signal-regulated protein kinase 2 (ERK2) but not c-Jun N-terminal kinase 1. tBHQ and SUL also activated MAPK kinase. Inhibition of MAPK kinase with its inhibitor, PD98059, abolished ERK2 activation and impaired the induction of quinone reductase, a phase II detoxifying enzyme, and antioxidant response element (ARE)-linked reporter gene by tBHQ and SUL. Overexpression of a dominant-negative mutant of ERK2 also attenuated tBHQ and SUL induction of ARE reporter gene activity. Interestingly, although expression of Ras and its mutant forms showed distinct effects on basal ARE reporter gene activity, they did not affect the activation of reporter gene by the inducers. Furthermore, a dominant-negative mutant of Ras had little effect on ERK2 activation by tBHQ and SUL, implicating a Ras-independent mechanism. Indeed, both tBHQ and SUL were able to stimulate Raf-1 kinase activity in vivo as well as in vitro. Thus, our results indicate that the induction of ARE-dependent phase II detoxifying enzymes is mediated by a MAPK pathway, which may involve direct activation of Raf-1 by the inducers.     

Mechanisms of regulating the Raf kinase family

The MAP Kinase pathway is a key signalling mechanism that regulates many cellular functions such as cell growth, transformation and apoptosis. One of the essential components of this pathway is the serine/threonine kinase, Raf. Raf (MAPKK kinase, MAPKKK) relays the extracellular signal from the receptor/Ras complex to a cascade of cytosolic kinases by phosphorylating and activating MAPK/ERK kinase (MEK; MAPK kinase, MAPKK) that phosphorylates and activates extracellular signal regulated kinase (ERK; mitogen-activated protein kinase, MAPK), which phosphorylates various cytoplasmic and nuclear proteins. Regulation of both Ras and Raf is crucial in the proper maintenance of cell growth as oncogenic mutations in these genes lead to high transforming activity. Ras is mutated in 30% of all human cancers and B-Raf is mutated in 60% of malignant melanomas. The mechanisms that regulate the small GTPase Ras as well as the downstream kinases MEK and extracellular signal regulated kinase (ERK) are well understood. However, the regulation of Raf is complex and involves the integration of other signalling pathways as well as intramolecular interactions, phosphorylation, dephosphorylation and protein-protein interactions. From studies using mammalian isoforms of Raf, as well as C. elegans lin45-Raf, common patterns and unique differences of regulation have emerged. This review will summarize recent findings on the regulation of Raf kinase.     

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.     

Urokinase receptor and fibronectin regulate the ERK(MAPK) to p38(MAPK) activity ratios that determine carcinoma cell proliferation or dormancy in vivo

We discovered that a shift between the state of tumorigenicity and dormancy in human carcinoma (HEp3) is attained through regulation of the balance between two classical mitogen-activated protein kinase (MAPK)-signaling pathways, the mitogenic extracellular regulated kinase (ERK) and the apoptotic/growth suppressive stress-activated protein kinase 2 (p38(MAPK)), and that urokinase plasminogen activator receptor (uPAR) is an important regulator of these events. This is a novel function for uPAR whereby, when expressed at high level, it enters into frequent, activating interactions with the alpha5beta1-integrin, which facilitates the formation of insoluble fibronectin (FN) fibrils. Activation of alpha5beta1-integrin by uPAR generates persistently high level of active ERK necessary for tumor growth in vivo. Our results show that ERK activation is generated through a convergence of two pathways: a positive signal through uPAR-activated alpha5beta1, which activates ERK, and a signal generated by the presence of FN fibrils that suppresses p38 activity. When fibrils are removed or their assembly is blocked, p38 activity increases. Low uPAR derivatives of HEp3 cells, which are growth arrested (dormant) in vivo, have a high p38/ERK activity ratio, but in spite of a similar level of alpha5beta1-integrin, they do not assemble FN fibrils. However, when p38 activity is inhibited by pharmacological (SB203580) or genetic (dominant negative-p38) approaches, their ERK becomes activated, uPAR is overexpressed, alpha5beta1-integrins are activated, and dormancy is interrupted. Restoration of these properties in dormant cells can be mimicked by a direct re-expression of uPAR through transfection with a uPAR-coding plasmid. We conclude that overexpression of uPAR and its interaction with the integrin are responsible for generating two feedback loops; one increases the ERK activity that feeds back by increasing the expression of uPAR. The second loop, through the presence of FN fibrils, suppresses p38 activity, further increasing ERK activity. Together these results indicate that uPAR and its interaction with the integrin should be considered important targets for induction of tumor dormancy.     

Extracellular signal regulated kinase 5 (ERK5) is required for the differentiation of muscle cells

Extracellular signal regulated kinase 5 (ERK5) is a novel member of the mitogen-activated protein kinase (MAPK) family with a poorly defined physiological function. Since ERK5 and its upstream activator MEK5 are abundant in skeletal muscle we examined a function of the cascade during muscle differentiation. We show that ERK5 is activated upon induction of differentiation in mouse myoblasts and that selective activation of the pathway results in promoter activation of differentiation-specific genes. Moreover, myogenic differentiation is completely blocked when ERK5 expression is inhibited by antisense RNA. Thus, we conclude that the MEK5/ERK5 MAP kinase cascade is critical for early steps of muscle cell differentiation.     

CK2 is acting upstream of MEK3/6 as a part of the signal control of ERK1/2 and p38 MAPK during keratinocytes autocrine differentiation

Protein kinase CK2 (formerly termed "casein kinase II") is a ubiquitously in mammalian cells distributed Ser/Thr kinase, with global role in cell regulation. Although, the involvement of CK2 in cell signalling is vast-investigated, virtually nothing is known about its contribution to signal control of keratinocytes differentiation. Here we show that, in autocrine differentiating keratinocytes, inhibition of the CK2 activity induced by 4,5,6,7-tetrabromobenzotriazole (TBB) causes reciprocal changes in the activities of major signal transduction regulators of keratinocytes differentiation, i.e. ERK1/2 and p38 MAPK, without affecting their protein levels. The ERK1/2 activity is strongly suppressed, while the activity of p38 is increased. We have also found that the activity of upstream and specific for p38 MAPK kinase MEK3/6 is also stimulated by TBB. These original results clearly demonstrate the participation of CK2 in the signal transduction pathway controlling MEK3/6, p38 MAPK, and ERK1/2 in the used model system.