Chow SY, Yu CY, Guy GR.
The Sprouty (Spry) proteins act as inhibitors of the Ras/ERK pathway downstream of receptor tyrosine kinases. In this study, we report a novel interaction between protein kinase C delta (PKCdelta) and Spry2. Endogenous PKCdelta and Spry2 interact in cells upon basic fibroblast growth factor stimulation, indicating a physiological relevance for the interaction. This interaction appeared to require the full-length Spry2 protein and was conformation-dependent. Conformational constraints were released upon FGFR1 activation, allowing the interaction to occur. Although this interaction did not affect the phosphorylation of PKCdelta by another kinase, it reduced the phosphorylation of a PKCdelta substrate, protein kinase D1 (PKD1). Spry2 was found to interact more strongly with PKCdelta with increasing amounts of PKD1, which indicated that instead of competing with PKD1 for binding with PKCdelta, it was more likely to form a trimeric complex with both PKCdelta and PKD1. Formation of the complex was found to be dependent on an existing PKCdelta-PKD1 interaction. By disrupting the interaction between PKCdelta and PKD1, Spry2 was unable to associate with PKCdelta to form the trimeric complex. As a consequence of this trimeric complex, the existing interaction between PKCdelta and PKD1 was increased, and the transfer of phosphate groups from PKCdelta to PKD1 was at least partly blocked by Spry2. The action of Spry2 on PKCdelta resulted in the inhibition of both ERK phosphorylation and invasion of PC-3 cells via PKCdelta signaling. By disrupting the capacity of PKCdelta to phosphorylate its cognate substrates, Spry2 may serve to modulate PKCdelta signaling downstream of receptor tyrosine kinases and to regulate the physiological outcome.
(A) Sprouty2 blocks PKCd from phosphorylating its substrate PKD1. Wild-type or Y55F mutant of Spry2 was co-expressed in 293T cells with either endogenous PKCd or myc-tagged wild-type PKCd, and FGFR1. Cell lysates were IP using PKD1 antibody, and the immunoprecipitates were immunoblotted with the antibodies shown on the left. WCL were also immunoblotted to confirm the presence of equal levels of proteins.
(B) Spry2 interacts with PKCδ and PKD1 to block phosphorylation of PKD1 by PKCδ. A summary schematic diagram showing the possible mechanism of interaction between Spry2, PKCδ and PKD1, and the domains involved: C2 (C2 domain on PKCδ), FYGMYD (the C2 recognition motif on PKD1), K376 (ATP binding site on PKCδ), T505 (phosphorylation site within the activation loop of PKCδ), S738 and S742 (phosphorylation sites within the activation loop of PKD1).
Published in J Biol Chem. 2009 Jul 17;284(29):19623-36.
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