Identification of insulin‐stimulated protein kinase‐1 as the rabbit equivalent of rsk^mo‐2: Identification of two threonines phosphorylated during activation by mitogen‐activated protein kinase

An improved procedure has been developed for the isolation of insulin‐stimulated protein kinase‐1 (ISPK‐1), an S6 kinase‐II homologue, by which 0.5 mg highly purified enzyme can be obtained within four days. The sequences of tryptic peptides from ISPK‐1 (100 residues) revealed 100% identity with the predicted protein product of rsk^mo‐2, a cDNA clone isolated from a mouse F2 cell line library [Alcorta, D. A., Crews, C. M., Sweet, L. J., Bankston, L., Jones, S. W. and Erikson, R. L. (1989) Mol. Cell. Biol. 9, 3850–3859], demonstrating that rsk^mo‐2 encodes an S6 kinase‐II. Two isoforms of mitogen‐activated protein (MAP) kinase (p42^mapk and p44^mapk) were the only ISPK‐1‐reactivating enzymes detected after Mono Q chromatography of extracts prepared from rabbit skeletal muscle or phaeochromocytoma 12 cells stimulated by nerve or epidermal growth factors. One of the residues on ISPK‐1 phosphorylated by p42^mapk was a threonine located nine residues N‐terminal to the conserved Ala‐Pro‐Glu motif in the C‐terminal protein kinase domain, an analogous location to phosphorylation sites essential for the activity of cAMP‐dependent protein kinase, MAP kinase and p34cdc2. A further threonine located five residues N‐terminal to the same Ala‐Pro‐Glu motif was also phosphorylated, probably via autophosphorylation catalysed by ISPK‐1 itself.