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Regulation of mammalian cell growth and proliferation is governed through receptor-mediated signaling networks that ultimately converge on the cell cycle machinery. Adaptor proteins play essential roles in the formation of intracellular signaling complexes, relaying extracellular signals from the plasma membrane to the nucleus of a cell. The leukocyte-specific adaptor protein Grap2 is a central linker protein in immune cell signaling and activation. Using Grap2 as bait protein, we identified a novel human protein, GCIP (Grap2 cyclin-D interacting protein). We found that GCIP bound to Grap2 in both yeast two-hybrid assays and in mammalian cells through binding to the COOH-terminal unique domain and SH3 domain (designated QC domain) of Grap2. GCIP also associated with cyclin D both in vitro and in vivo. The expression of GCIP was found in all human tissues examined with the highest level of expression in the heart, muscle, peripheral blood leukocytes, and brain. Furthermore, phosphorylation of retinoblastoma protein by cyclin D-dependent protein kinase was reduced and E2F1-mediated transcription activity was inhibited in cells transfected with GCIP. High level expression of GCIP in terminally differentiated tissues and the inhibition of E2F1 transcription activation suggest that GCIP could play an important role in controlling cell differentiation and proliferation.

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Using degenerate oligonucleotide primers derived from conserved regions in the catalytic domains of protein kinases, we have identified transcripts of the protein kinase families in Trypanosoma brucei by the polymerase chain reaction technique. From the cDNAs synthesized from poly(A)+ RNA purified from the bloodstream form of the pathogen, we have obtained seven distinct partial cDNA sequences. Deduced amino acid sequences of these seven clones contain conserved regions characteristic of catalytic domains of eukaryotic protein serine/threonine kinases. DNA gel blots showed that one of the clones, TbPK-A4 is most likely a member of a subfamily in the protein kinase gene family, whereas the other six are probably each encoded by a single gene in the genome of T. brucei. The full-length cDNA of TbPK-A1 was cloned, sequenced, and found to encode an open reading frame of 350 amino acid residues. Its gene (designated KFR1) demonstrated high sequence similarity to KSS1 and FUS3 from Saccharomyces cerevisiae and rat MAP kinase at the amino acid level. There are a 3- to 4-fold higher level of KFR1 transcript and a 2-fold increase of KFR1 protein in the bloodstream form when compared with the insect form of T. brucei. This preferential expression of KFR1 in the bloodstream form of T. brucei may play a role in controlling the cell cycle and thus the growth rate of the organism.