CHARACTERIZATION OF BRAIN PCTAIRE-1 KINASE IMMUNOREACTIVITY AND ITS INTERACTIONS WITH P11 AND 14-3-3 PROTEINS

An antibody directed against the C-terminal part of PCTAIRE-1 recogniz ed three proteins ill rodent brain. The high-molecular-mass band is mo st abundant in the cerebellum, hippocampus and cortex. It migrated at the same apparent molecular mass as recombinant PCTAIRE-1 and interact ed. like recombinant PCTAIRE-1, with p11 and 14-3-3 proteins Combinati on of p11 or 14-3-3 affinity resins with immunoprecipitation and pepti de elution allowed us to obtain a purified full-length PCTAIRE-1 prepa ration having significant kinase activity, These results suggest that PCTAIRE-1 is an active kinase in brain. The catalytic core region of P CTAIRE-1 which is common for all cyclin-dependent kinases, does not in teract with pll and 14-3-3 proteins in the two-hybrid assay, Full inte raction with pll and 14-3-3 proteins requires both, the N-terminal and C-terminal ends of PCTAIRE-1. suggesting that complex three-dimension al arrangements art, responsible for these interactions, A low-molecul ar-mass protein (migrating at about 30 kDa) that was also recognized b y the antibody directed against the carboxy-terminal part of PCTAIRE-1 , is abundant and almost homogeneously distributed in all brain areas investigated, Database searches starting with the amino acid sequences of two peptides obtained by tryptic digestion of this protein yielded cDNA and genomic (a gene of about 10 kb on human chromosome 1q24-1q25 and clone 262D12) sequences, allowing us to compose a DNA sequence co ding for a putative 26 kDa protein containing both peptides. This prot ein has no important sequence similarity with ally other known protein , But many DNA sequences are found in databases with an almost 100% id entity with parts of the 26 kDa protein coding sequence. Our results a llow us to attribute these widely distributed cDNA sequences to an exi sting 26-kDa protein and to localize a gene within two recently publis hed genomic sequences.