Abstract 1436: The Cdk5 activator p39 specifically links muskelin to myosin and regulates stress fiber organization and contraction

Abstract

Abstract Cyclin-dependent kinase 5 (Cdk5), a proline-directed serine/threonine protein kinase, regulates signaling pathways implicated in the adhesion and migration of various cell types. Activation of Cdk5 requires its association with its activators p35 or p39. We have recently shown, in lens epithelial cells, that Cdk5 kinase activity regulates rho-dependent myosin phosphorylation, cytoskeletal contraction, and cell migration by controlling Src-p190RhoGAP signaling. In a separate line of investigation, we demonstrated that the N-terminus of p39 binds myosin essential light chain (MLC), while the C-terminus of p39 binds muskelin, a multi-domain scaffolding protein. As both muskelin and MLC have been associated with cytoskeletal organization, the present study seeks to determine whether p39 might serve as an adaptor that links muskelin to myosin and to investigate the possible biological relevance of such an interaction. Immunoprecipitation studies demonstrated that muskelin forms a protein complex with p39, MLC, myosin regulatory light chain (MRLC), and myosin heavy chain II (MHC II). Suppressing expression of p39 abrogated the interaction between muskelin and the myosin subunits, demonstrating that p39 is required to link muskelin to myosin. Immunofluorescence staining showed that muskelin colocalized with MRLC and actin-containing stress fibers. Suppressing muskelin expression with either of two different siRNAs significantly reduced MRLC phosphorylation and disrupted stress fiber organization. Similar effects were observed when Cdk5 activity was inhibited or suppressed. Moreover, suppressing muskelin expression prevented any further reduction of MRLC phosphorylation when Cdk5 activity was blocked, indicating that muskelin plays an important role in Cdk5-dependent regulation of MRLC phosphorylation. The Cdk5 activating protein p39 was necessary and sufficient for Cdk5-dependent regulation of myosin phosphorylation, as suppression of p39, but not p35, significantly reduced MRLC phosphorylation. Together, these results demonstrate that the Cdk5 activating protein p39 specifically links muskelin to myosin and thus, to stress fibers. Therefore, p39 has a dual role in cytoskeletal regulation: on one hand, it is an integral subunit of Cdk5 kinase; on the other, it acts as an adaptor protein to recruit muskelin to stress fibers. Each of these p39 functions is required for myosin phosphorylation, cytoskeletal contraction, and efficient cell migration. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1436. doi:10.1158/1538-7445.AM2011-1436