Previous studies from this laboratory identified a 28-kd nonreducible
protein, liver-derived immunoinhibitory factor (LDIF) from the mouse liver. Isolation of this
protein resulted in the co-purification of another unique
protein called heat responsive
protein 12 kd (Hrp12). In contrast to LDIF, Hrp12 was totally reducible to a
protein of 12 kd suggesting a dimer.
Sodium dodecyl sulfate-
polyacrylamide gel electrophoresis (SDS-PAGE) purification, followed by sequencing of an in situ
cyanogen bromide digest of membrane bound Hrp12, yielded an internal 20-amino
acid polypeptide. Degenerate
oligonucleotides made from this
peptide were used to screen a murine liver
complementary DNA (cDNA) library. A 1240-bp
cDNA clone was obtained with an internal 521-bp open reading 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 perchloric acid soluble rat
protein (PSP). Transcripts for Hrp12 were mainly restricted to the liver and kidney in mouse and man. The
protein was estimated to be approximately 0.8% of the total liver-soluble cytosolic
protein. A zoo-blot probed at moderate stringency with labeled
cDNA revealed a strong conservation of the gene in all of the mammalian species tested. Analysis of the
protein structure of Hrp12 revealed motifs predicted to be targets for
protein kinase C (PKC). More importantly, purified mouse Hrp12 could be phosphorylated in vitro with PKC. The
protein had significant similarity to DnaK
heat shock protein (Hsp)70 and contained a 54-amino
acid stretch with sequence similarity to Hsp90. This prompted us to investigate the heat shock response of Hrp12. Isolated hepatocytes and
hepatoma cells were exposed to different heat 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 out. The message for this novel
protein responded atypically to heat shock. Although the steady-state level of the message increased after heat shock, a marked oscillatory pattern was superimposed on it. In contrast, the steady-state levels of Hsp90 and Hsp70
messenger RNA (
mRNA) were found to respond to heat shock in the expected manner. Finally, the amount of
Hrp12 protein was also found to increase after heat shock in a manner that was consistent with heat-responsive
proteins.