Phosphorylation of ribosomal protein S6 at multiple sites by a cyclic AMP-independent protein kinase from lymphoid cells.

Ribosomes prepared from murine lymphosarcoma cells were phosphorylated by a cyclic AMP-independent protein kinase designated H4P kinase. H4P kinase was isolated as an inactive enzyme which was activated by Mg2+-ATP and an endogenous converting enzyme. In the absence of preactivation by Mg2+-ATP and an endogenous converting enzyme, H4P kinase catalyzed phosphorylation of 80, 60, and 40 S ribosomal subunits at a low rate. After activation, the H4P kinase selectively catalyzed phosphorylation of the S 6 protein in the 40 S ribosomal subunit. Under the assay conditions selected, at least 90% of the [32P]phosphate transferred to the 40 S ribosomal preparation was incorporated into S 6. The apparent Km for 40 S subunits phosphorylated by H4P kinase was 7.2 microM. The calculated Vmax was 50 nmol of Pi transferred per min/mg. Exhaustive phosphorylation of 40 S subunits resulted in incorporation of 3 mol of phosphate/mol of S 6, in contrast to results reported previously which indicated 0.3 mol of phosphate was transferred by a similar enzyme from reticulocyte (Del Grande, R. W., and Traugh, J. A. (1982) Eur. J. Biochem. 123, 421-428). These data are consistent with a potential role for H4P kinase in the insulin-mediated phosphorylation of S 6 at multiple sites.