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 reading frame encodes a 45 kDa protein containing 32% proline. To begin characterizing the CR16 protein, a rabbit polyclonal antibody was raised against an Escherchia coli-produced fusion protein containing amino acids 370-438 of CR16. The antibody identifies a protein doublet of 68 and 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, when hippocampal extracts are electrophoresed on nondenaturing polyacrylamide gels, the CR16 protein migrates as a 48 kDa protein that better correlates 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. In addition, CR16 has at least 36 copies of the PXXP motif, which is contained in all known SH3 binding domains. Solution and filter binding assays confirm that CR16 selectively binds SH3 domains. The CR16 primary amino acid sequence also contains at least eight consensus MAP kinase phosphorylation sites, five of which are in the potential SH3 binding domains. The CR16 protein, immunoprecipitated from rat brain, is an in vitro substrate for the purified enzyme. However, phosphorylation of CR16 does not greatly affect the binding of the various SH3 domains in our 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.
Department of Cell Biology, Neurobiology and Anatomy, Ohio State Biochemistry Program, Ohio State University, Columbus, USA.
M C
MC
Weiler
J L
JL
Smith
J N
JN
Masters
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D001665:Binding Sites; D001921:Brain / Q000378:metabolism*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D003001:Cloning, Molecular; D016384:Consensus Sequence; D003598:Cytoskeletal Proteins; D018076:DNA, Complementary; D008297:Male; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism*; D016366:Open Reading Frames; D010452:Peptide Biosynthesis; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D010750:Phosphoproteins; D055232:Proline-Rich Protein Domains; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D018909:src Homology Domains
D000906:Antibodies; D003598:Cytoskeletal Proteins; D018076:DNA, Complementary; D009419:Nerve Tissue Proteins; D010455:Peptides; D010750:Phosphoproteins; D011994:Recombinant Proteins; C105364:Wipf3 protein, rat; D017871:Calcium-Calmodulin-Dependent Protein Kinases