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 purified 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 frame, which did not correspond to any known protein, was predicted to have 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 kinases. Northern blot analysis indicated that HASPP28 was present in all rat tissues tested, including those from the brain, lung, spleen, kidney, liver, heart, and muscle, in decreasing order of abundance. Phosphopeptide analysis of rHASPP28 phosphorylated in vitro by various kinases showed different tryptic patterns on two-dimensional mapping and isoelectric focusing gels. From [32P]PO4-labeled N1E115 neuroblastoma cells, HASPP28 can be immunoprecipitated with a polyclonal antiserum 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 treated with phorbol 12-myristate 13-acetate or 8-bromo-cAMP did not show any increased phosphorylation over those of untreated ones, and the phosphopeptide patterns of the immunoprecipitates again were similar to that of CK II phosphorylated protein. These results suggest that HASPP28 is a novel phosphoprotein that can be phosphorylated by several kinases in vitro. In intact cells, CK II seems to be solely responsible for the phosphorylation of HASPP28.
Shen
L
L
Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA.
Huang
K P
KP
Chen
H C
HC
Huang
F L
FL
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D001921:Brain / Q000378:metabolism*; D047390:Casein Kinase II; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D015723:Gene Library; D015151:Immunoblotting; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D016366:Open Reading Frames; D009928:Organ Specificity; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010449:Peptide Mapping; D010750:Phosphoproteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D013379:Substrate Specificity
D017931:DNA Primers; D018076:DNA, Complementary; D009419:Nerve Tissue Proteins; C098600:Pdap1 protein, rat; D010446:Peptide Fragments; D010750:Phosphoproteins; D011994:Recombinant Proteins; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases