Abstract
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.
Highlights
In response to extracellular mitogenic and growth signals, cells initiate different signaling pathways that lead to the transcriptional activation of downstream genes and cell proliferation
Molecular Cloning of Human GCIP—To identify proteins that interact with human Grap2, the COOH-terminal unique glutamine-/proline-rich domain and the SH3 domain (QC domain) of Grap2 was used as a bait to screen a pretransformed human bone marrow cDNA library using the yeast two-hybrid system
We have cloned a novel human protein from bone marrow library, GCIP, that bound to the leukocyte-specific adaptor protein Grap2 and the key cell cycle protein, cyclin D
Summary
Retinoblastoma protein; SH, Src homology domain; RT-PCR, reverse transcriptase-polymerase chain reaction; HA, hemagglutinin; GST, glutathione S-transferase; CAT, chloramphenicol acetyltransferase; HLH, helix-loop-helix; Cdk, cyclin Ddependent protein kinase; PAGE, polyacrylamide gel electrophoresis. Overexpression of GCIP in mammalian cells suppressed the E2F1-mediated transcriptional activity, which is required for the transition of the G1 phase to the S phase in cell cycle progression Together these data suggest that GCIP is a novel human protein potentially involved in the regulation of cell differentiation and proliferation through Grap and cyclin D-mediated signaling pathways
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