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Identification of a novel cyclin-like protein in human tumor cells
R T Williams1, L Wu, D A Carbonaro-Hall
1Division of Orthopaedic Surgery, Childrens Hospital Los Angeles, University of Southern California School of Medicine.
Abstract:
Cyclins are key regulatory proteins that, in concert with cyclin-dependent protein kinase subunits (cdks), function to govern critical transitions and/or restriction points during the course of cell cycle progression. Recently, a number of putative mammalian G1 cyclins have been characterized at the molecular level; however, the specific activities of the cyclin/cdk complexes and the precise biochemical pathways regulated by the G1 cyclins remain to be elucidated. In the present study we identify a novel cyclin-like protein in pediatric bone and extremity tumors that appears to be related to, but is clearly distinct from, previously identified members of the cyclin D family, as determined by its profile of antibody cross-reactivity, apparent molecular size, chromatographic behavior, physicochemical properties, and pattern of peptide mapping. This 46-kDa cyclin-like protein, tentatively designated p46cyclin X, is first expressed in synchronized MG-63 osteosarcoma cells in mid-G1, well after the induction of p36cyclin D1, yet prior to the induction of cyclins E and A. Northern analysis, utilizing an oligonucleotide probe complementary to an epitope shared by cyclins D1, D2, and X, detected a novel mRNA species, the appearance of which correlates with p46cyclin X expression. The p46cyclin X protein in Ewing's sarcomas and Wilms' tumors is electrophoretically and chromatographically distinct from both p36cyclin D1 and p34cyclin D2. Moreover, the p46cyclin X protein is 1) precipitated by p9Ckshs1-agarose beads, 2) physically associated with p33cdk2, and 3) autophosphorylated in in vitro kinase reactions. Taken together with the biochemical data, the temporal expression of the p46cyclin X/p33cdk2 kinase system is suggestive of a potential role in regulating latter G1 events (i.e. START) in the commitment to S phase.
Insights
A novel cyclin-like protein, p46cyclin X, was identified in pediatric tumors. This protein, distinct from cyclin D family members, associates with CDK2 and may regulate late G1 cell cycle events.
Area of Science:
- Molecular Biology
- Cell Cycle Regulation
- Oncology
Background:
- Cyclins and cyclin-dependent kinases (cdks) regulate cell cycle transitions.
- While G1 cyclins are known, their specific roles and regulated pathways require further elucidation.
- Novel G1 cyclins are crucial for understanding cell cycle control.
Purpose of the Study:
- To identify and characterize a novel cyclin-like protein in pediatric bone and extremity tumors.
- To determine the molecular properties and expression patterns of this new protein.
- To investigate its potential role in cell cycle regulation.
Main Methods:
- Protein characterization using antibody cross-reactivity, molecular size, chromatography, and peptide mapping.
- Analysis of protein expression timing in synchronized MG-63 osteosarcoma cells.
- Northern analysis to detect corresponding mRNA species.
- Biochemical assays including precipitation, association with cdk2, and in vitro kinase activity.
Main Results:
- A novel 46-kDa cyclin-like protein, p46cyclin X, was identified in pediatric tumors.
- p46cyclin X is distinct from known cyclin D family members (D1, D2).
- Expression of p46cyclin X occurs in mid-G1, preceding cyclins E and A.
- p46cyclin X physically associates with p33cdk2 and exhibits autophosphorylation.
- A novel mRNA species correlates with p46cyclin X expression.
Conclusions:
- p46cyclin X represents a novel cyclin distinct from the cyclin D family.
- The p46cyclin X/p33cdk2 kinase system is expressed in late G1.
- This system potentially regulates late G1 events, including commitment to S phase.