Different carboxyl-terminal domain kinase activities are induced by heat-shock and arsenite. Characterization of their substrate specificity, separation by Mono Q chromatography, and comparison with the mitogen-activated protein kinases.
In response to heat-shock and chemical treatments, cells undergo profound biochemical changes such as modifications in protein phosphorylation in order to resist the new, unfavorable growth conditions. We have previously shown that in HeLa cells a protein kinase (HS-CTD kinase) activity is induced rapidly after a heat or sodium arsenite shock. This kinase activity is able to phosphorylate a synthetic peptide composed of four repeats of the motif Ser-Pro-Thr-Ser-Pro-Ser-Tyr, a motif highly repeated in the carboxyl-terminal domain (CTD) of the largest subunit of eukaryotic RNA polymerase II. In this paper, we designed a new experimental procedure to characterize the substrate specificity of this kinase activity. We show that HS-CTD kinase activity phosphorylates a consensus sequence (-P-X-S/T-P-) which is similar to the sequence phosphorylated by extracellular regulated protein kinases (also called mitogen-activated protein kinases). However, there is a slight but reproducible difference between these kinases in their use of serine or threonine as the phosphate acceptor. Mono Q chromatography allows the separation of five stress-induced CTD kinase activities, two of which coelute with active mitogen-activated protein kinase forms revealed by Western blotting with anti ERK1-ERK2 antibodies. The other three CTD kinase activities induced after a stress are distinct from ERK1 and ERK2 and have different enzymatic properties. The molecular nature of these HS-CTD kinases and the physiological significance of their activation during stress remain to be determined.
Trigon
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Unité de Génétique moléculaire, Ecole Normale Superieure, Paris, France.
Morange
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D000595:Amino Acid Sequence; D018053:Arsenites / Q000494:pharmacology*; D002852:Chromatography, Ion Exchange; D016384:Consensus Sequence; D004790:Enzyme Induction; D006367:HeLa Cells; D006358:Hot Temperature; D006801:Humans; D008969:Molecular Sequence Data; D011494:Protein Kinases / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D013379:Substrate Specificity
D018053:Arsenites; D011494:Protein Kinases; C060137:carboxy-terminal domain kinase