Sprouty2 Interacts with Protein Kinase Cδ and Disrupts Phosphorylation of Protein Kinase D1

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 δ (PKCδ) and Spry2. Endogenous PKCδ 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 PKCδ by another kinase, it reduced the phosphorylation of a PKCδ substrate, protein kinase D1 (PKD1). Spry2 was found to interact more strongly with PKCδ with increasing amounts of PKD1, which indicated that instead of competing with PKD1 for binding with PKCδ, it was more likely to form a trimeric complex with both PKCδ and PKD1. Formation of the complex was found to be dependent on an existing PKCδ-PKD1 interaction. By disrupting the interaction between PKCδ and PKD1, Spry2 was unable to associate with PKCδ to form the trimeric complex. As a consequence of this trimeric complex, the existing interaction between PKCδ and PKD1 was increased, and the transfer of phosphate groups from PKCδ to PKD1 was at least partly blocked by Spry2. The action of Spry2 on PKCδ resulted in the inhibition of both ERK phosphorylation and invasion of PC-3 cells via PKCδ signaling. By disrupting the capacity of PKCδ to phosphorylate its cognate substrates, Spry2 may serve to modulate PKCδ signaling downstream of receptor tyrosine kinases and to regulate the physiological outcome.