Abstract

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The level of p67 gradually increases when mouse C2C12 myoblasts differentiate into myotubes. During this differentiation, cycling myoblasts permanently withdraw from cell cycle. Largely, the cyclin/Cdk complexes drive eukaryotic cell cycle. To investigate whether p67 has any role(s) while C2C12 myoblasts are withdrawing from the cell cycle, we examined the levels of cyclins and Cdks in these myoblasts constitutively expressing rat p67 and its mutants D251A, H231E, D6/2, and K1K2. We found that p67 is involved in lowering the overall concentration of cyclin D1/Cdk4 and cyclin D1/Cdk6 complexes and thus inhibits the cell cycle at the G₁-phase. It is also involved in possible proteolytic processing of pro-forms of cyclins E, A, and B1 but not cyclin D1 to maturation. P67 may be involved in lowering the level of Cdk2 possibly by proteolytic degradation and increasing the level of Cdk1 by an unknown mechanism. P67 is involved in the increased level of cyclin A possibly to show positive outcome during DNA synthesis. It also showed strong effects on degradation of cyclin B1 possibly due to their direct or indirect binding - indicating p67’s involvement in the inhibition of G₂-phase and G₂/M-phase transition. We also observed that when p67’s auto-proteolysis activity was increased due to the change of H231 residue to E231, this mutant showed dominating effect over endogenous p67. Altogether, our data indicate that increased level of p67 during myoblasts’ differentiation into myotube is possibly to inhibit cell cycle at the G₁-phase, G₁/S-phase transition, G₂-phase, and G₂/M-phase transition.

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