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Cloning and characterization of murine p16INK4a and p15INK4b genes
D E Quelle1, R A Ashmun, G J Hannon
1Howard Hughes Medical Institute, Memphis, Tennessee, USA.
Abstract:
Progression through the G1 phase of the cell cycle is regulated in part by the D-type cyclin-dependent kinases, cdk4 and cdk6. Genes encoding two specific inhibitors of these kinases, human p16(INK4a/MTS1) and p15(INK4b/MTS2), map to a region of common cytogenetic abnormalities on chromosome 9p21. The murine cognates of these genes were isolated and identified as mouse p16INK4a and p15INK4b based on their homology to their human counterparts and their selective transcriptional induction by SV40T-antigen and TGF-beta, respectively. Both genes map to position C3-C6 on mouse chromosome 4, in a region syntenic with human chromosome 9p. Amplification of polyadenylated mRNA by polymerase chain reactions revealed no expression of mouse p16INK4a in many normal tissues, whereas p15INK4b was expressed ubiquitously. Like human p16INK4a, mouse p16INK4a binds specifically to cdk4 and cdk6 in vitro and inhibits the phosphorylation of the retinoblastoma protein, pRb, by each of these cyclin D-dependent kinases. In mouse MEL erythroleukemia cells, p16INK4a associates preferentially with cdk6 under conditions where cdk4 and cdk6 are coexpressed at equivalent levels. Expression vectors encoding human or mouse p16INK4a caused G1 phase arrest in NIH3T3 fibroblasts, and cyclin D1- and cdk4-dependent pRb kinase activities were inhibited in the p16INK4a-arrested cells.
Insights
Mouse p16INK4a inhibits cell cycle progression by binding to cyclin-dependent kinases (cdk4 and cdk6). This study identifies mouse p16INK4a and p15INK4b, revealing conserved functions with human counterparts in cell cycle regulation.
Area of Science:
- Cell Biology
- Molecular Biology
- Cancer Research
Background:
- Cell cycle progression, particularly through G1 phase, is crucial for cell proliferation.
- D-type cyclin-dependent kinases (cdk4/cdk6) are key regulators of the G1 phase.
- Human p16INK4a and p15INK4b are inhibitors of cdk4/cdk6 and map to chromosome 9p21, a region with frequent abnormalities.
Purpose of the Study:
- To isolate and characterize the murine homologs of human p16INK4a and p15INK4b.
- To investigate the expression patterns and biochemical functions of mouse p16INK4a and p15INK4b.
- To determine the role of mouse p16INK4a in cell cycle regulation.
Main Methods:
- Gene isolation and identification based on homology and transcriptional induction.
- Polymerase chain reaction (PCR) for mRNA expression analysis.
- In vitro binding assays and kinase activity inhibition studies.
- Expression vector transfection and cell cycle analysis (G1 arrest).
Main Results:
- Mouse p16INK4a and p15INK4b were identified and mapped to mouse chromosome 4, syntenic with human 9p.
- Mouse p16INK4a showed limited expression in normal tissues, while p15INK4b was ubiquitous.
- Mouse p16INK4a specifically bound cdk4/cdk6, inhibited retinoblastoma protein (pRb) phosphorylation, and caused G1 arrest in fibroblasts.
- p16INK4a preferentially associated with cdk6 in mouse erythroleukemia cells.
Conclusions:
- Mouse p16INK4a and p15INK4b are functional homologs of their human counterparts.
- Mouse p16INK4a acts as a specific inhibitor of cdk4/cdk6, regulating cell cycle progression at G1.
- These findings highlight the conserved role of p16INK4a in cell cycle control across species.