Association of atypical protein kinase C isotypes with the Docker protein FRS2 in fibroblast growth factor signaling

Abstract

FRS2 is a docker protein that recruits signaling proteins to the plasma membrane in fibroblast growth factor signal transduction. We report here that FRS2 was associated with PKC λ when Swiss 3T3 cells were stimulated with basic fibroblast growth factor. PKC ζ, the other member of the atypical PKC subfamily, could also bind FRS2. The association between FRS2 and PKC λ is likely to be direct as shown by yeast two-hybrid analysis. The C-terminal fragments of FRS2 (amino acid residues 300-508) and SNT2 (amino acids 281- 492), an isoform bearing 50% identity to FRS2, interacted with PKC A at a region (amino acids 240-562) that encompasses the catalytic domain. In vitro kinase assays revealed neither FRS2 nor SNT2 was a substrate of PKC λ or ζ. Mutation of the alanine residue (Ala-120) to glutamate in the pseudo- substrate region of PKC λ results in a constitutively active kinase that exhibited more than 2-fold greater binding to FRS2 in vitro than its 'closed' wild-type counterpart. Tyrosine phosphorylation of FRS2 did not affect its binding to the constitutively active PKC λ mutant, suggesting that the activation of PKC λ is necessary and sufficient for its association with FRS2. It is likely that FRS2 serves as an anchoring protein for targeting activated atypical PKCs to the cell plasma membrane in signaling pathways.