This is a request to analyze the mechanism of cell cycle regulation by cell adhesion. The applicant has found that cell adhesion regulates G1 cell cycle progression at multiple steps. Specifically, cell adhesion regulates the transcription of cyclins A and D, the translation of cyclin D, and the activations of cyclins D/cdk4/6 and cyclin E/cdk2 kinase activities. He now proposes to extend these studies by a detailed analysis of the effects of cell adhesion on the cyclin D1 promoter. These analyses will include the mutational analysis of the relevant promoter structure, an analysis of biosynthesis of specific CRE binding proteins. The latter is motivated by preliminary data that suggests that the CRE in the cyclin D1 promoter is critical for adhesion regulated transcription. Secondly, the effect of cell adhesion on the translation of cyclin D1 will be examined. Specifically the applicant hypothesizes that PHAS-I dephosphorylation results in its binding to the eIF-4e to prevent formation of initiation complex. His further hypothesis, that this complex is specifically required for the translation of mRNA's with stem loop structures in their five prime untranslated region. A computer analysis of cyclin D1 has identified such a potential stem loop structure. Thus, he will examine the effects of cell adhesion on the phosphorylation of PHAS-I and eIF-4e levels and determine their association by co-precipitation. In addition, studies are proposed to analyze the regions in the five prime untranslated region of the cyclin D mRNA that may be involved in efficiency of translation.
A third aim will address the requirement of adhesion for activation of cyclin D/cdk4/6 activity. In particular, the effect of cell adhesion on the formation of this complex will be assessed in effort to ascertain whether the inhibition is mediated by INK4 or CKI family inhibitors. Depending on which family is involved, efforts will be undertaken to discern whether specific INK4 or CKI family members show adhesion dependent expression. Other studies will examine potential effects of cell adhesion of phosphorylation of CDK4. Integrins are known to activate the ERK1/2 pathway. The applicant has preliminary data that ectopic activation of this pathway can partially rescue the cyclin D1 expression defect in suspended cells. More detailed experiments are proposed to analyze the capacity of constitutive activation of MAP kinase pathway, to overcome the various adhesion defects in the cell cycle kinases.
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