A kinetic approach towards understanding substrate interactions and the catalytic mechanism of the serine/threonine protein kinase ERK2: identifying a potential regulatory role for divalent magnesium.
We are interested in the mechanism and regulation of the extracellular regulated protein kinases, ERK1 and ERK2, due to their key roles in cellular signal transduction and disease. Both enzymes phosphorylate a large number of structurally disparate proteins upon activation by phorbol esters, serum and growth factors, and are activated through a protein kinase cascade, termed the mitogen activated protein kinase (MAPK) pathway. ERK2 catalyses the transfer of the gamma-phosphate of adenosine triphosphate to serine or threonine residues found in Ser-Pro or Thr-Pro motifs on proteins. Its catalytic mechanism is intriguing, because it appears to predominantly rely on interactions outside of the active site cleft to specify a substrate. To study ERK2, we developed a recombinant protein called EtsDelta138, which comprises residues 1-138 of the transcription factor Ets-1, an excellent substrate of ERK2. Here we review several steady-state kinetic experiments that reveal details of the ERK2 mechanism and a hitherto unknown process of ERK2 activation by free magnesium. The physiological relevance of this mechanism is discussed.
Waas
William F
WF
Division of Medicinal Chemistry, College of Pharmacy, University of Texas at Austin, Austin, TX 78712, USA.
Rainey
Mark A
MA
Szafranska
Anna E
AE
Cox
Kari
K
Dalby
Kevin N
KN
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D002413:Cations, Divalent / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism*; D019950:Mitogen-Activated Protein Kinase 1 / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050784:Proto-Oncogene Protein c-ets-1; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D050783:Proto-Oncogene Proteins c-ets; D015398:Signal Transduction; D013379:Substrate Specificity; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
D002413:Cations, Divalent; C494041:ETS1 protein, human; D050784:Proto-Oncogene Protein c-ets-1; D011518:Proto-Oncogene Proteins; D050783:Proto-Oncogene Proteins c-ets; D014157:Transcription Factors; D000255:Adenosine Triphosphate; D019950:Mitogen-Activated Protein Kinase 1; D008274:Magnesium