HRP12, A NOVEL HEAT-RESPONSIVE, TISSUE-SPECIFIC, PHOSPHORYLATED PROTEIN ISOLATED FROM MOUSE-LIVER

Previous studies from this laboratory identified a 28-kd nonreducible protein, liver-derived immunoinhibitory factor (LDIF) from the mouse l iver, Isolation of this protein resulted in the co-purification of ano ther unique protein called heat responsive protein 12 kd (Hrp12), In c ontrast to LDIF, Hrp12 was totally reducible to a protein of 12 kd sug gesting a dimer. Sodium dodecyl sulfate-polyacrylamide gel electrophor esis (SDS-PAGE) purification, followed by sequencing of an in situ cya nogen bromide digest of membrane bound Hrp12, yielded an internal 20-a mino acid polypeptide. Degenerate oligonucleotides made from this pept ide were used to screen a murine liver complementary DNA (cDNA) librar y, A 1240-bp cDNA clone was obtained with an internal 521-bp open read ing frame (ORF). Sequence analysis of the 173-amino acid ORF of mouse Hrp12 showed a high degree of homology with a 99 amino acid rat liver- kidney perchloric acid-soluble protein (LKPS) and a 136-amino acid per chloric acid soluble rat protein (PSP), Transcripts for Hrp12 were mai nly restricted to the liver and kidney in mouse and man, The protein w as estimated to be approximately 0.8% of the total liver-soluble cytos olic protein, A zoo-blot probed at moderate stringency with labeled cD NA revealed a strong conservation of the gene in all of the mammalian species tested. Analysis of the protein structure of Hrp12 revealed mo ths predicted to be targets for protein kinase C (PKC). More important ly, purified mouse Hrp12 could be phosphorylated in vitro with PKC, Th e protein had significant similarity to DnaK heat shock protein (Hsp)7 0 and contained a 54-amino acid stretch with sequence similarity to Hs p90. This prompted us to investigate the heat shock response of Hrp12. Isolated hepatocytes and hepatoma cells were exposed to different hea t shock temperatures (39.5 degrees C, 42.5 degrees C, and 44.5 degrees C); and then total RNA was extracted and Northern analysis carried ou t, The message for this novel protein responded atypically to heat sho ck. Although the steady state level of the message increased after hea t shock, a marked oscillatory pattern was superimposed on it, In contr ast, the steady-state levels of Hsp90 and Hsp70 messenger RNA (mRNA) w ere found to respond to heat shock in the expected manner. Finally, th e amount of Hrp12 protein was also found to increase after heat shock in a manner that was consistent with heat-responsive proteins.