CR16, A NOVEL PROLINE-RICH PROTEIN EXPRESSED IN RAT-BRAIN NEURONS, BINDS TO SH3 DOMAINS AND IS A MAP KINASE SUBSTRATE

CR16 is a glucocorticoid-regulated gene expressed in subpopulations of neurons in the brain, including the hippocampus. The CR16 open readin g frame encodes a 45 kDa protein containing 32% proline. To begin char acterizing the CR16 protein, a rabbit polyclonal antibody was raised a gainst an Escherchia coil-produced fusion protein containing amino aci ds 370-438 of CR16. The antibody identifies a protein doublet of 68 an d 72 kDa by sodium-dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) from hippocampal extracts and from insect cells expressing the CR16 open reading frame from a baculovirus construct. However, whe n hippocampal extracts are electrophoresed on nondenaturing polyacryla mide gels, the CR16 protein migrates as a 48 kDa protein that better c orrelates with the size of the open reading frame. Examination of the primary amino acid sequence reveals at least 12 sequence homologies to the abl-SH3 binding domain consensus sequence XPXXPPP Psi XP. Ln addi tion, CR16 has at least 36 copies of the PXXP motif, which is containe d in all known SH3 binding domains. Solution and filter binding assays confirm that CR16 selectively binds SH3 domains. The CR16 primary ami no acid sequence also contains at least eight consensus MAP kinase pho sphorylation sites, five of which are in the potential SH3 binding dom ains. The CR16 protein, immunoprecipitated from rat brain, is an in vi tro substrate for the purified enzyme. However, phosphorylation of CR1 6 does not greatly affect the binding of the various SH3 domains in ou r assay system. These data strongly suggest that the function of CR16 is to mediate one or more signal transduction pathways in CNS neurons, in addition to being a glucocorticoid-regulated gene.