PubMed · 9149529
The source did not provide an abstract. Follow the original record for more information.
Explore connections, maps & timelines
L Holm, C Sander. 1997. Enzyme HIT.. https://doi.org/10.1016/s0968-0004(97)01021-9
Cite the original work for its findings. Save a collection to share your selection of sources.
KEEP EXPLORING
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.
BACKGROUND: The origins of and interrelations between low grade and high grade neuroendocrine lung tumors, typical and atypical carcinoids, and small cell lung carcinoma (SCLC) have not been elucidated. Karyotypic and molecular genetic studies have demonstrated deletions in 3p in 100% of SCLCs and the candidate lung tumor suppressor gene, FHIT, at 3p14.2 is not expressed in the majority of SCLCs. Similar studies of typical and atypical carcinoids could clarify the interrelations among these tumors. METHODS: For molecular genetic analyses, archival carcinoids and paired normal cells were microdissected from paraffin sections, deparaffinized, and DNA prepared. Oligonucleotide primer pairs for 12 microsatellite markers mapping between 3p14.2 and 3p21.3 were used to amplify allelic DNA fragments from 13 typical and 6 atypical carcinoids. In addition, an independent series of archival sections of carcinoids and SCLCs was tested by immunohistochemistry for expression of Fhit protein. RESULTS: Of the six atypical carcinoids examined, three had lost an allele at all informative markers, whereas one had lost alleles in two distinct regions and two showed allele loss in a subregion of the chromosome region tested. Of the 13 typical carcinoids, 3 showed allele loss at only 1 or 2 loci each. Typical carcinoids, similar to normal lung epithelia, were strongly positive for the cytoplasmic Fhit protein, SCLCs were uniformly negative, and atypical carcinoids appeared to express an intermediate level of Fhit protein. CONCLUSIONS: Loss of heterozygosity at 3p14.2-p21.3 is significantly more extensive in all atypical carcinoids. Atypical carcinoids, which exhibit clinicopathologic features intermediate between typical carcinoids and small cell carcinomas and have been considered well differentiated neuroendocrine carcinomas, also are intermediate between typical carcinoids and SCLC on the basis of extent of loss of 3p alleles and reduced expression of Fhit protein.
The amino acid sequence of the Saccharomyces cerevisiae mRNA 5'-triphosphatase (TPase) diverges from those of higher eukaryotes. In order to confirm the sequence divergence of TPases in lower and higher eukaryotes, the Candida albicans gene for TPase was identified and characterized. This gene designated CaCET1 (C. albicans mRNA 5'-capping enzyme triphosphatase 1) has an open reading frame of 1.5 kb, which can encode a 59-kDa protein. Although the N-terminal one-fifth of S. cerevisiae TPase (ScCet1p) is missing in CaCet1p, CaCet1p shares significant sequence similarity with ScCet1p over the entire region of the protein; the recombinant CaCet1p, which was expressed as a fusion protein with glutathione S-transferase (GST), displayed TPase activity in vitro. CaCET1 rescued CET1-deficient S. cerevisiae cells when expressed under the control of the ADH1 promoter, whereas the human capping enzyme derivatives that are active for TPase activity but defective in mRNA 5'-guanylyltransferase (GTase) activity did not. Yeast two-hybrid analysis revealed that C. albicans Cet1p can bind to the S. cerevisiae GTase in addition to its own partner, the C. albicans GTase. In contrast, neither the full-length human capping enzyme nor its TPase domain interacted with the yeast GTase. These results indicate that the failure of the human TPase activity to complement an S. cerevisiae cet1delta null mutation is attributable, at least in part, to the inability of the human capping enzyme to associate with the yeast GTase, and that the physical association of GTase and TPase is essential for the function of the capping enzyme in vivo.