A NOVEL SRC-SUPPRESSED AND RAS-SUPPRESSED PROTEIN-KINASE-C SUBSTRATE ASSOCIATED WITH CYTOSKELETAL ARCHITECTURE

We previously identified a novel src- and ras-suppressed gene, 322, en coding a mitogenic regulatory function (Lin, X., Nelson, P. J., Frankf ort, B., Tombler, E., Johnson, R., and Gelman, I. H. (1995) Mol. Cell. Biol. 15, 2754-2762). Here, we characterize the 322 gene product as a n in vivo and in vitro substrate of protein kinase C (PKC). Hence, we named this product SSeCKS (pronounced essex) for Src Suppressed C Kina se Substrate. Rabbit polyclonal sera raised against glutathione S-tran sferase (GST)-SSeCKS recognized a myristylated 280/290-kDa doublet in Rat-6 fibroblasts. SSeCKS levels in src- and ras-transformed Rat-6 cel ls were 15- and 8-fold less, respectively, than those in untransformed cells. Short-term addition of phorbol ester resulted in a Ei-fold inc rease in SSeCKS phosphorylation a which was inhibited by bis-indolylma leimide. In vitro phosphorylation of GST-SSeCKS by purified rabbit bra in PKC-alpha was enhanced by phosphatidylserine and blocked by excess PKC pseudosubstrate inhibitor peptide. GST-SSeCKS bound purified PKC-a lpha or PKC from Rat-6 lysates in a phosphatidylserine-dependent manne r. Four SSeCKS domains containing Lys/Arg-rich moths similar to the PH C phosphorylation site in MARCKS were phosphorylated in vitro by PKC. Immunofluorescence analysis showed SSeCKS present throughout the cytop lasm with enrichment in podosomes and at the cell edge. Short-term add ition of phorbol esters caused the movement of SSeCKS from plasma memb rane sites to the perinucleus coincident with a loss of actin stress f ibers. These data suggest a role for SSeCKS in the control of cellular cytoskeletal architecture.