Characterization of PKCI and comparative studies with FHIT, related members of the HIT protein family.
We previously described the isolation of a human cDNA that encodes a protein termed protein kinase C inhibitor (hPKCI). We elucidated the three-dimensional structure of this protein and demonstrated that in vitro, it enzymatically hydrolyzes adenosine polyphosphates. To identify other proteins that interact with hPKCI, in the present study, we used the hPKCI as a bait in the yeast two-hybrid system, together with a mouse embryo cDNA library. This led to the isolation of a murine PKCI homologue (mPKCI). This finding is consistent with our previous structural studies indicating that hPKCI exists as a homodimer and indicates the strong conservation of the PKCI sequence during evolution. Northern blot analysis indicated that a 0.7-kb PKCI mRNA was expressed in several tissues obtained from adult mice and also in a variety of rodent and human cell lines. Western blot analyses, using a polyclonal antibody prepared against hPKCI, indicated that this protein is expressed at relatively high levels in several murine tissues and in a variety of human cell lines prepared from normal tissues or tumors. In contrast to these findings, parallel studies with a polyclonal antibody to FHIT, a related histidine triad (HIT) protein and putative tumor suppressor, indicated that FHIT was expressed at low or undetectable levels in some of the same cell lines. Microscopy of immunostained cells indicated that the PKCI protein was present mainly in the nucleus of both normal and tumor-derived epithelial cell lines. Evidence presented in this and previous studies suggest that in vivo the ubiquitously expressed PKCI protein does not function as an inhibitor of PKC but rather acts as an enzyme in a yet to be identified pathway.
Herbert Irving Comprehensive Cancer Center, Room 1509, 701 West 168th Street, New York, New York, 10032, USA.
M G
MG
Klein
Y
Y
Yao
E D
ED
Slosberg
C D
CD
Lima
Y
Y
Doki
I B
IB
Weinstein
D017766:Acid Anhydride Hydrolases; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000302:isolation & purification; D006801:Humans; D006867:Hydrolases; D007424:Intracellular Fluid / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D005810:Multigene Family; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics; D009928:Organ Specificity / Q000235:genetics; D011506:Proteins / Q000737:chemistry* / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D014407:Tumor Cells, Cultured; D000099170:Bis(5'-adenosyl)triphosphatase
D017766:Acid Anhydride Hydrolases; D018076:DNA, Complementary; D006867:Hydrolases; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D011506:Proteins; D000099170:Bis(5'-adenosyl)triphosphatase; C063306:HINT1 protein, human; C489275:Hint1 protein, mouse; C097552:histidine triad protein