Functional expression and growth factor activation of an epitope-tagged p44 mitogen-activated protein kinase, p44mapk.
Mitogen-activated protein kinases (MAPKs) or extracellular signal-regulated kinases (ERKs) are serine/threonine kinases of apparent Mr 42-44 kDa that are rapidly activated by a variety of extracellular signals in many cell types. This activation coincides with their phosphorylation on tyrosine and threonine residues, and these covalent modifications are required for full activity of the enzymes. They are thought to play a pivotal role in integrating and transmitting transmembrane signals for growth and differentiation. Here, we report the cloning, sequence, and functional expression in fibroblasts of the hamster p44 MAP kinase (p44mapk). The protein deduced from the nucleotide sequence of an almost full-length cDNA is 98.6% homologous to the rat p44mapk (ERK1). To distinguish the expression of the cloned cDNA from the endogenous p44mapk, we fused to the 5' end of the cDNA an initiating codon followed by an influenza hemagglutinin 9-residue peptide epitope (HAP). The chimeric kinase HAP/p44mapk, under transcriptional control of the cytomegalovirus promoter, was stably expressed in Chinese hamster lung fibroblasts in a functional form. We show that its basal activity, measured by phosphorylation of the substrate myelin basic protein, is activated severalfold (up to 25) by the mitogens alpha-thrombin, platelet-derived growth factor, and fetal calf serum. In addition, we report that in response to alpha-thrombin, this activation is rapid (6-fold in 1 min), biphasic (first peak at 5 min, second broader peak at 1-2 h), persistent (for greater than or equal to 4 h), and parallel to an increased phosphorylation on tyrosine.We conclude that the constructed and stably expressed chimera, HAP/p44mapk, has retained apparently all the hormonal regulation features of the endogenous form. This system now offers the possibility to study structure-function relationships and to determine the role of this kinase in growth control.
Centre de Biochimie-CNRS, Université de Nice, France.
S
S
Meloche
G
G
Pagès
J
J
Pouysségur
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D004789:Enzyme Activation; D000939:Epitopes; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D015870:Gene Expression; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D008934:Mitogens / Q000494:pharmacology; D008969:Molecular Sequence Data; D010766:Phosphorylation; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D011494:Protein Kinases / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013917:Thrombin / Q000494:pharmacology; D014162:Transfection; D014443:Tyrosine / Q000737:chemistry
D000939:Epitopes; D008934:Mitogens; D010982:Platelet-Derived Growth Factor; D011993:Recombinant Fusion Proteins; D016222:Fibroblast Growth Factor 2; D014443:Tyrosine; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D013917:Thrombin