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.