MOLECULAR-CLONING AND CHARACTERIZATION OF A NOVEL CASEIN KINASE-II SUBSTRATE, HASPP28, FROM RAT-BRAIN

HASPP28 (heat- and acid-stable phosphoprotein of 28 kDa) has been puri fied to near homogeneity from the acid-stable protein fraction of rat brain extract, Based on the N-terminal 40 amino acid sequence, a pair of highly degenerate primers was used to generate a 107-bp probe from rat brain RNA by RT-PCR, From the rat brain lambda gt11 library, this probe identified two positive clones that together provided a cDNA of 837 bp with an open reading frame of 546 bp. This cDNA was extended by 3 'RACE to 1.2 kb that included a polyadenylation signal and a poly(A ) tail. The 180-amino-acid sequence derived from the open reading fram e, which did not correspond to any known protein, was predicted to hav e phosphorylation sites for protein kinase C, casein kinase II (CKII), and protein kinase A. Indeed, both the purified rat brain HASPP28 and the recombinant HASPP28 (rHASPP28) can be phosphorylated by these kin ases, Northern blot analysis indicated that HASPP28 was present in all rat tissues tested, including those from the brain, lung, spleen, kid ney, liver, heart, and muscle, in decreasing order of abundance. Phosp hopeptide analysis of rHASPP28 phosphorylated in vitro by various kina ses showed different tryptic peptide patterns on two-dimensional mappi ng and isoelectric focusing gels, From [P-32]PO4-labeled N1E115 neurob lastoma cells, HASPP28 can be immunoprecipitated with a polyclonal ant iserum raised against rHASPP28. The immunoprecipitated protein showed a phosphopeptide pattern similar to that of rHASPP28 phosphorylated by CK II in vitro. Furthermore, the immunoprecipitates from cells treate d with phorbol 12-myristate 13-acetate or 8-bromo-cAMP did not show an y increased phosphorylation over those of untreated ones, and the phos phopeptide patterns of the immunoprecipitates again were similar to th at of CK II phosphorylated protein. These results suggest that HASPP28 is a novel phosphoprotein that can be phosphorylated by several kinas es in vitro. In intact cells, CK II seems to be solely responsible for the phosphorylation of HASPP28. (C) 1996 Academic Press, Inc.