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pRb-Dependent Cyclin D3 Protein Stabilization Is Required for Myogenic Differentiation ^,

Francesca De Santa, Sonia Albini, Eleonora Mezzaroma, Livio Baron, Armando Felsani,^* and Maurizia Caruso^*

CNR-Istituto di Neurobiologia e Medicina Molecolare, Fondazione S. Lucia, Via Fosso di Fiorano, 64-00143 Roma, Italy

Received 23 November 2006/ Returned for modification 29 December 2006/ Accepted 7 August 2007

The expression of retinoblastoma (pRb) and cyclin D3 proteins is highly induced during the process of skeletal myoblast differentiation. We have previously shown that cyclin D3 is nearly totally associated with hypophosphorylated pRb in differentiated myotubes, whereas Rb^–/– myocytes fail to accumulate the cyclin D3 protein despite normal induction of cyclin D3 mRNA. Here we report that pRb promotes cyclin D3 protein accumulation in differentiating myoblasts by preventing cyclin D3 degradation. We show that cyclin D3 displays rapid turnover in proliferating myoblasts, which is positively regulated through glycogen synthase kinase 3ß (GSK-3ß)-mediated phosphorylation of cyclin D3 on Thr-283. We describe a novel interaction between pRb and cyclin D3 that maps to the C terminus of pRb and to a region of cyclin D3 proximal to the Thr-283 residue and provide evidence that the pRb-cyclin D3 complex formation in terminally differentiated myotubes hinders the access of GSK-3ß to cyclin D3, thus inhibiting Thr-283 phosphorylation. Interestingly, we observed that the ectopic expression of a stabilized cyclin D3 mutant in C2 myoblasts enhances muscle-specific gene expression; conversely, cyclin D3-null embryonic fibroblasts display impaired MyoD-induced myogenic differentiation. These results indicate that the pRb-dependent accumulation of cyclin D3 is functionally relevant to the process of skeletal muscle cell differentiation.

Published ahead of print on 20 August 2007.

We dedicate this work to the memory of Cesare Vesco.

Present address: Department of Experimental Oncology, European Institute of Oncology, Via Ripamonti, 435-20141 Milano, Italy.

Present address: Research Center Ospedale Bambino Gesù, Piazza Sant' Onofrio 4-00165, Roma, Italy.