14-3-3 proteins mediate interactions between proteins involved in signal transduction and cell cycle regulation. Phosphorylation of target proteins as well as 14-3-3 are important for protein-protein interactions. Here, we describe the purification of a protein kinase from porcine brain that phosphorylates 14-3-3 on Thr-233. This protein kinase has been identified as casein kinase I␣ (CKI␣) by peptide mapping analysis and sequencing. Among mammalian 14-3-3, only 14-3-3 possesses a

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... le residue at the same position (Ser-233), and we show that this residue is also phosphorylated by CKI. In addition, we show that 14-3-3 is exclusively phosphorylated on Thr-233 in human embryonic kidney 293 cells. The residue 233 is located within a region shown to be important for the association of 14-3-3 to target proteins. We showed previously that, in 293 cells, only the unphosphorylated form of 14-3-3 associates with the regulatory domain of c-Raf. We have now shown that in vivo phosphorylation of 14-3-3 at the CKI␣ site (Thr-233) negatively regulates its binding to c-Raf, and may be important in Raf-mediated signal transduction.