A novel src- and ras-suppressed protein kinase C substrate associated with cytoskeletal architecture.
We previously identified a novel src- and ras-suppressed gene, 322, encoding a mitogenic regulatory function (Lin, X., Nelson, P. J., Frankfort, B., Tombler, E., Johnson, R., and Gelman, I. H. (1995) Mol. Cell. Biol. 15, 2754-2762). Here, we characterize the 322 gene product as an in vivo and in vitro substrate of protein kinase C (PKC). Hence, we named this product SSeCKS (pronounced essex) for Src Suppressed C Kinase Substrate. Rabbit polyclonal sera raised against glutathione S-transferase (GST)-SSeCKS recognized a myristylated 280/290-kDa doublet in Rat-6 fibroblasts. SSeCKS levels in src- and ras-transformed Rat-6 cells were 15- and 8-fold less, respectively, than those in untransformed cells. Short-term addition of phorbol ester resulted in a 5-fold increase in SSeCKS phosphorylation which was inhibited by bis-indolylmaleimide. In vitro phosphorylation of GST-SSeCKS by purified rabbit brain PKC-alpha was enhanced by phosphatidylserine and blocked by excess PKC pseudosubstrate inhibitor peptide. GST-SSeCKS bound purified PKC-alpha or PKC from Rat-6 lysates in a phosphatidylserine-dependent manner. Four SSeCKS domains containing Lys/Arg-rich motifs similar to the PKC phosphorylation site in MARCKS were phosphorylated in vitro by PKC. Immunofluorescence analysis showed SSeCKS present throughout the cytoplasm with enrichment in podosomes and at the cell edge. Short-term addition of phorbol esters caused the movement of SSeCKS from plasma membrane sites to the perinucleus coincident with a loss of actin stress fibers. These data suggest a role for SSeCKS in the control of cellular cytoskeletal architecture.
Lin
X
X
Department of Microbiology, Mount Sinai School of Medicine, New York, New York 10029, USA. igelman@smtplink.mssm.edu
Tombler
E
E
Nelson
P J
PJ
Ross
M
M
Gelman
I H
IH
D016475:3T3 Cells; D054758:A Kinase Anchor Proteins; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D018797:Cell Cycle Proteins; D002461:Cell Line, Transformed; D002645:Chickens; D003599:Cytoskeleton / Q000737:chemistry*; D005982:Glutathione Transferase / Q000378:metabolism; D006358:Hot Temperature; D051379:Mice; D008934:Mitogens / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames; D010766:Phosphorylation; D011489:Protein Denaturation; D011493:Protein Kinase C / Q000378:metabolism*; D011817:Rabbits; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D054758:A Kinase Anchor Proteins; C491161:Akap12 protein, mouse; C102361:Akap12 protein, rat; D018797:Cell Cycle Proteins; D008934:Mitogens; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D011493:Protein Kinase C