A proline-rich sequence unique to MEK1 and MEK2 is required for raf binding and regulates MEK function

Mammalian MEK1 and MEK2 contain a proline-rich (PR) sequence that is absent both from the yeast homologs Ste7 and Byr1 and from a recently cloned activator of the JNK/stress-activated protein kinases, SEK1/MKK4. Since this PR sequence occurs in MEKs that are regulated by Raf family enzymes but is missing from MEKs and SEKs activated independently of Raf, we sought to investigate the role of this sequence in MEK1 and MEK2 regulation and function. Deletion of the PR sequence from MEK1 blocked the ability of MEK1 to associate with members of the Raf family and markedly attenuated activation of the protein in vivo following growth factor stimulation. In addition, this sequence was necessary for efficient activation of MEK1 in vitro by B-Raf but dispensable for activation by a novel MEK1 activator which we have previously detected in fractionated fibroblast extracts. Furthermore, we found that a phosphorylation site within the PR sequence of MEK1 was required for sustained MEK1 activity in response to serum stimulation of quiescent fibroblasts. Consistent with this observation, we observed that MEK2, which lacks a phosphorylation site at the corresponding position, was activated only transiently following serum stimulation. Finally, we found that deletion of the PR sequence from a constitutively activated MEK1 mutant rendered the protein nontransforming in Rat1 fibroblasts. These observations indicate a critical role for the PR sequence in directing specific protein-protein interactions important for the activation, inactivation, and downstream functioning of the MEKs.

[1]  K. Guan,et al.  Nerve growth factor stimulates a novel protein kinase in PC-12 cells that phosphorylates and activates mitogen-activated protein kinase kinase (MEK). , 1995, Biochemical Journal.

[2]  M. Weber,et al.  Biochemical Analysis of MEK Activation in NIH3T3 Fibroblasts , 1995, The Journal of Biological Chemistry.

[3]  Jiahuai Han,et al.  Independent human MAP-kinase signal transduction pathways defined by MEK and MKK isoforms , 1995, Science.

[4]  D. Baltimore,et al.  Modular binding domains in signal transduction proteins , 1995, Cell.

[5]  C. Marshall,et al.  Specificity of receptor tyrosine kinase signaling: Transient versus sustained extracellular signal-regulated kinase activation , 1995, Cell.

[6]  L. Zon,et al.  Role of SAPK/ERK kinase-1 in the stress-activated pathway regulating transcription factor c-Jun , 1994, Nature.

[7]  L. Zon,et al.  Activation of stress-activated protein kinase by MEKK1 phosphorylation of its activator SEK1 , 1994, Nature.

[8]  G L Johnson,et al.  Differential activation of ERK and JNK mitogen-activated protein kinases by Raf-1 and MEKK. , 1994, Science.

[9]  M. Weber,et al.  RAS and RAF-1 form a signalling complex with MEK-1 but not MEK-2 , 1994, Molecular and cellular biology.

[10]  M. Weber,et al.  Partial purification of a mitogen-activated protein kinase kinase activator from bovine brain. Identification as B-Raf or a B-Raf-associated activity. , 1994, The Journal of biological chemistry.

[11]  W. Kolch,et al.  Association of MEK1 with p21ras.GMPPNP is dependent on B-Raf , 1994, Molecular and cellular biology.

[12]  R. Jove,et al.  Expression, purification and characterization of recombinant mitogen-activated protein kinase kinases. , 1994, The Biochemical journal.

[13]  G. Johnson,et al.  B-Raf-dependent regulation of the MEK-1/mitogen-activated protein kinase pathway in PC12 cells and regulation by cyclic AMP , 1994, Molecular and cellular biology.

[14]  G. Landreth,et al.  The mitogen-activated protein kinase cascade is activated by B-Raf in response to nerve growth factor through interaction with p21ras , 1994, Molecular and cellular biology.

[15]  Michel Morange,et al.  A novel kinase cascade triggered by stress and heat shock that stimulates MAPKAP kinase-2 and phosphorylation of the small heat shock proteins , 1994, Cell.

[16]  R. Erikson,et al.  Constitutive activation of Mek1 by mutation of serine phosphorylation sites. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[17]  N. Ahn,et al.  Transformation of mammalian cells by constitutively active MAP kinase kinase. , 1994, Science.

[18]  R. Davis,et al.  An osmosensing signal transduction pathway in mammalian cells. , 1994, Science.

[19]  L Bibbs,et al.  A MAP kinase targeted by endotoxin and hyperosmolarity in mammalian cells. , 1994, Science.

[20]  J. W. Gloor,et al.  Mitogen-activated protein (MAP) kinase phosphorylation of MAP kinase kinase: determination of phosphorylation sites by mass spectrometry and site-directed mutagenesis. , 1994, Journal of biochemistry.

[21]  D. Templeton,et al.  Identification of 2 serine residues of MEK-1 that are differentially phosphorylated during activation by raf and MEK kinase. , 1994, The Journal of biological chemistry.

[22]  A. Brunet,et al.  Constitutive mutant and putative regulatory serine phosphorylation site of mammalian MAP kinase kinase (MEK1). , 1994, The EMBO journal.

[23]  C. Marshall,et al.  Activation of MAP kinase kinase is necessary and sufficient for PC12 differentiation and for transformation of NIH 3T3 cells , 1994, Cell.

[24]  A. Brunet,et al.  Growth factor‐stimulated MAP kinase induces rapid retrophosphorylation and inhibition of MAP kinase kinase (MEK1) , 1994, FEBS letters.

[25]  J. Hancock,et al.  Activation of Raf as a result of recruitment to the plasma membrane. , 1994, Science.

[26]  Sally J. Leevers,et al.  Requirement for Ras in Raf activation is overcome by targeting Raf to the plasma membrane , 1994, Nature.

[27]  J. Woodgett,et al.  The stress-activated protein kinase subfamily of c-Jun kinases , 1994, Nature.

[28]  T. Haystead,et al.  Insulin activates a novel adipocyte mitogen-activated protein kinase kinase kinase that shows rapid phasic kinetics and is distinct from c-Raf. , 1994, The Journal of biological chemistry.

[29]  A. Ashworth,et al.  Identification of the sites in MAP kinase kinase‐1 phosphorylated by p74raf‐1. , 1994, The EMBO journal.

[30]  M. Karin,et al.  JNK1: A protein kinase stimulated by UV light and Ha-Ras that binds and phosphorylates the c-Jun activation domain , 1994, Cell.

[31]  Philip R. Cohen,et al.  The threonine residues in MAP kinase kinase 1 phosphorylated by MAP kinase in vitro are also phosphorylated in nerve growth factor‐stimulated rat phaeochromocytoma (PC12) cells , 1994, FEBS letters.

[32]  P. Dent,et al.  Mitogen-activated protein kinase kinase 1 (MKK1) is negatively regulated by threonine phosphorylation , 1994, Molecular and cellular biology.

[33]  C. Zheng,et al.  Activation of MEK family kinases requires phosphorylation of two conserved Ser/Thr residues. , 1994, The EMBO journal.

[34]  J. Avruch,et al.  Enzymatic characteristics of the c-Raf-1 protein kinase. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[35]  C. Crews,et al.  Raf-1 forms a stable complex with Mek1 and activates Mek1 by serine phosphorylation. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[36]  R. Medema,et al.  Epidermal growth factor induces phosphorylation of extracellular signal-regulated kinase 2 via multiple pathways , 1993, Molecular and cellular biology.

[37]  J. Bishop,et al.  Association of pRas and pRaf-1 in a complex correlates with activation of a signal transduction pathway , 1993, Current Biology.

[38]  F. McCormick,et al.  Reconstitution of the Raf-1—MEK—ERK Signal Transduction Pathway In Vitro , 1993, Molecular and cellular biology.

[39]  M. McMahon,et al.  Conditional transformation of cells and rapid activation of the mitogen-activated protein kinase cascade by an estradiol-dependent human raf-1 protein kinase , 1993, Molecular and cellular biology.

[40]  C. Marshall,et al.  A dominant-negative mutant of raf blocks mitogen-activated protein kinase activation by growth factors and oncogenic p21ras. , 1993, The Journal of biological chemistry.

[41]  P. Dent,et al.  Identification and characterization of a new mammalian mitogen-activated protein kinase kinase, MKK2 , 1993, Molecular and cellular biology.

[42]  C. Crews,et al.  Extracellular signals and reversible protein phosphorylation: What to Mek of it all , 1993, Cell.

[43]  J. Avruch,et al.  Mitogen regulation of c-Raf-1 protein kinase activity toward mitogen-activated protein kinase-kinase. , 1993, The Journal of biological chemistry.

[44]  S. Elledge,et al.  Normal and oncogenic p21ras proteins bind to the amino-terminal regulatory domain of c-Raf-1 , 1993, Nature.

[45]  A. Ashworth,et al.  Complementation of byrl in fission yeast by mammalian MAP kinase kinase requires coexpression of Raf kinase , 1993, Nature.

[46]  P. Warne,et al.  Direct interaction of Ras and the amino-terminal region of Raf-1 in vitro , 1993, Nature.

[47]  Jonathan A. Cooper,et al.  Mammalian Ras interacts directly with the serine/threonine kinase raf , 1993, Cell.

[48]  R. Davis,et al.  The mitogen-activated protein kinase signal transduction pathway. , 1993, The Journal of biological chemistry.

[49]  M. Wigler,et al.  Complex formation between RAS and RAF and other protein kinases. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[50]  M. Weber,et al.  Complexes of Ras.GTP with Raf-1 and mitogen-activated protein kinase kinase. , 1993, Science.

[51]  K. Guan,et al.  Cloning and characterization of two distinct human extracellular signal-regulated kinase activator kinases, MEK1 and MEK2. , 1993, The Journal of biological chemistry.

[52]  Paul W. Sternberg,et al.  C. elegans lin-45 raf gene participates in let-60 ras-stimulated vulval differentiation , 1993, Nature.

[53]  N. Perrimon,et al.  Developmental and molecular characterization of mutations in the Drosophila-raf serine/threonine protein kinase. , 1993, Development.

[54]  C. Lange-Carter,et al.  A divergence in the MAP kinase regulatory network defined by MEK kinase and Raf , 1993, Science.

[55]  B. Errede,et al.  A conserved kinase cascade for MAP kinase activation in yeast. , 1993, Current opinion in cell biology.

[56]  Jonathan A. Cooper,et al.  Mos stimulates MAP kinase in Xenopus oocytes and activates a MAP kinase kinase in vitro , 1993, Molecular and cellular biology.

[57]  Y. Nishida,et al.  A protein kinase similar to MAP kinase activator acts downstream of the raf kinase in Drosophila , 1993, Cell.

[58]  E. Hafen,et al.  Raf functions downstream of Rasl in the Sevenless signal transduction pathway , 1992, Nature.

[59]  P. Cohen,et al.  Sustained activation of the mitogen-activated protein (MAP) kinase cascade may be required for differentiation of PC12 cells. Comparison of the effects of nerve growth factor and epidermal growth factor. , 1992, The Biochemical journal.

[60]  P. Cohen,et al.  Activation of the MAP kinase pathway by the protein kinase raf , 1992, Cell.

[61]  C. Crews,et al.  The primary structure of MEK, a protein kinase that phosphorylates the ERK gene product. , 1992, Science.

[62]  T. Haystead,et al.  Activation of mitogen-activated protein kinase kinase by v-Raf in NIH 3T3 cells and in vitro. , 1992, Science.

[63]  David L. Brautigan,et al.  Raf-1 activates MAP kinase-kinase , 1992, Nature.

[64]  J. Pouysségur,et al.  Coordinate, biphasic activation of p44 mitogen-activated protein kinase and S6 kinase by growth factors in hamster fibroblasts. Evidence for thrombin-induced signals different from phosphoinositide turnover and adenylylcyclase inhibition. , 1992, The Journal of biological chemistry.

[65]  L. Heasley,et al.  The beta-PDGF receptor induces neuronal differentiation of PC12 cells. , 1992, Molecular biology of the cell.

[66]  M. Cobb,et al.  Extracellular signal-regulated kinases: ERKs in progress. , 1991, Cell regulation.

[67]  P. Cohen,et al.  Dissection of the protein kinase cascade by which nerve growth factor activates MAP kinases , 1991, Nature.

[68]  J. Avruch,et al.  pp54 microtubule-associated protein-2 kinase requires both tyrosine and serine/threonine phosphorylation for activity. , 1991, The Journal of biological chemistry.

[69]  T. Sturgill,et al.  Recent progress in characterization of protein kinase cascades for phosphorylation of ribosomal protein S6. , 1991, Biochimica et biophysica acta.

[70]  J. Shabanowitz,et al.  Identification of the regulatory phosphorylation sites in pp42/mitogen‐activated protein kinase (MAP kinase). , 1991, The EMBO journal.

[71]  W. Kolch,et al.  Raf-1 protein kinase is required for growth of induced NIH/3T3 cells , 1991, Nature.

[72]  J. Maller,et al.  Requirement for integration of signals from two distinct phosphorylation pathways for activation of MAP kinase , 1990, Nature.

[73]  N. Perrimon,et al.  Requirement of the Drosophila raf homologue for torso function , 1989, Nature.

[74]  C. Der,et al.  Biological and biochemical properties of human ras H genes mutated at codon 61 , 1986, Cell.

[75]  Brian J. Stevenson,et al.  Functional homology of protein kinases required for sexual differentiation in Schizosaccharomyces pombe and Saccharomyces cerevisiae suggests a conserved signal transduction module in eukaryotic organisms. , 1993, Molecular biology of the cell.

[76]  L. Heasley The,B-PDGF Receptor Induces Neuronal , 1992 .

[77]  T. Hunter,et al.  Phosphopeptide mapping and phosphoamino acid analysis by two-dimensional separation on thin-layer cellulose plates. , 1991, Methods in enzymology.

[78]  A. Miller,et al.  Long-term expression of human adenosine deaminase in mice after transplantation of bone marrow infected with amphotropic retroviral vectors. , 1990, Human gene therapy.