Identification of Src as a novel atypical protein kinase C-interacting protein.
Atypical protein kinase C-zeta (PKC-zeta) participates in nerve growth factor (NGF) signaling and is required for NGF-induced differentiation of PC12 cells. The biological activity of PKC-zeta is likely mediated by interaction of PKC-zeta with specific proteins. Affinity column chromatography employing the PKC-zeta regulatory domain coupled to glutathione-agarose was used to search for proteins that bound PKC-zeta. Two proteins (59/60 kDa) were recovered from NGF-stimulated PC12 cell lysates that bound the matrix. Western blot analysis of pooled column fractions identified these proteins as tubulin and src, respectively. Using purified preparations of src and tubulin, PKC-zeta was shown to interact with both proteins using blot overlay. To demonstrate a functional interaction in vivo, PC12 cells expressing a temperature-sensitive v-src were shifted to the permissive temperature (37 degrees C), followed by immunoprecipitation. At the permissive temperature where src was active, PKC-zeta was tyrosine phosphorylated and coassociated with src in vivo; by comparison, at the nonpermissive temperature (40 degrees C) PKC-zeta was not tyrosine phosphorylated. Taken together, these findings support a novel role for the interaction of src and atypical PKC in vivo, which is dependent upon the activity of src and the tyrosine phosphorylation state of PKC-zeta.
Seibenhener
M L
ML
Department of Biological Sciences, Auburn University, Alabama 36849, USA.
Roehm
J
J
White
W O
WO
Neidigh
K B
KB
Vandenplas
M L
ML
Wooten
M W
MW
D000818:Animals; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D002846:Chromatography, Affinity; D015688:Oncogene Protein pp60(v-src) / Q000737:chemistry / Q000378:metabolism*; D016716:PC12 Cells; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D011493:Protein Kinase C / Q000737:chemistry / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D000099221:Protein Kinase C zeta
D000911:Antibodies, Monoclonal; D015688:Oncogene Protein pp60(v-src); D011493:Protein Kinase C; D011505:Protein-Tyrosine Kinases; D000099221:Protein Kinase C zeta