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Involvement of cdc2-mediated phosphorylation in the cell cycle-dependent regulation of p185neu
N Kiyokawa1, D Karunagaran, E K Lee
1Department of Tumor Biology, The University of Texas M.D. Anderson Cancer Center, Houston 77030, USA.
Abstract:
We previously reported cell cycle-dependent negative regulation of p185neu (decreased tyrosine phosphorylation and kinase activity, with electrophoretic mobility retarded by serine/threonine phosphorylation) in M phase and the escape of mutation-activated p185neu* from this regulation. Our present results showed that retardation of electrophoretic mobility occurs independently of the cells' transformed status. We found that normal p185neu lost its ability to dimerize in the M phase. We demonstrated a physical association between cdc2 (a serine/threonine kinase, active in M phase) and p185neu. We showed that the carboxy terminal portion of p185neu is phosphorylated in vitro by cdc2. Many phosphopeptides (at least three phosphoserine residues) unique to the M phase were identified, and the in vivo and in vitro phosphopeptide patterns were superimposable. In contrast, mutation-activated p185neu* dimerized in the M phase with no changes in electrophoretic mobility, failed to associate with cdc2 and no unique phosphoserine residues could be identified in the M phase (data not shown), consistent with the escape of p185neu* from cell cycle-dependent regulation. Our results suggest that this escape is an intrinsic property of the mutation-activated p185neu* independent of its ability to transform cells. Our results also suggest the involvement of serine/threonine kinases such as cdc2 in the cell cycle-dependent negative regulation of p185neu.
Insights
Normal p185neu is regulated by cell cycle kinases like cdc2 during M phase, but mutation-activated p185neu* escapes this regulation. This escape is an intrinsic property of mutated p185neu*, independent of cell transformation.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Cell cycle regulation of protein activity is crucial for normal cell function.
- p185neu, a receptor tyrosine kinase, undergoes cell cycle-dependent negative regulation in M phase.
- Mutation-activated p185neu* escapes this regulation, but the underlying mechanisms remain unclear.
Purpose of the Study:
- To investigate the mechanisms of cell cycle-dependent regulation of p185neu.
- To determine if the escape of mutation-activated p185neu* from regulation is linked to its transforming ability.
Main Methods:
- Analysis of p185neu dimerization and association with cdc2 during the cell cycle.
- In vitro and in vivo phosphopeptide mapping of p185neu.
- Comparison of normal p185neu and mutation-activated p185neu* behavior in M phase.
Main Results:
- Normal p185neu loses dimerization ability and associates with cdc2 in M phase, leading to serine/threonine phosphorylation.
- Mutation-activated p185neu* retains dimerization ability, does not associate with cdc2, and lacks M phase-specific phosphorylation.
- Electrophoretic mobility retardation of p185neu occurs independently of cellular transformation status.
Conclusions:
- The cell cycle-dependent negative regulation of p185neu involves serine/threonine kinases like cdc2.
- The escape of mutation-activated p185neu* from this regulation is an intrinsic property, not dependent on its transforming ability.