Protein phosphorylation by kinases plays a central role in the regulation and coordination of multiple biological processes. We have studied the substrate specificity of atypical human vaccinia-related kinases (VRK1 and VRK2) using a human peptide-arraycontaining 1080 sequences phosphorylated in known signaling pathways. We identified for VRK1 and VRK2 a subset of potential peptide targets, all of them result in a consensus sequence composed of at least four basic residues. One of these target proteins is coilin, which is the scaffold protein on which Cajal bodies are assembled in the nucleus. VRK1 phosphorylates coilin in Ser184. Coilin co-localizes and interacts with VRK1 in Cajal bodies (CB), but not with the mutant VRK1 (R358X) that causes pombocerebelar degeneration and spinal muscular atrophy. VRK1 (R358X) is less active than VRK1. VRK1 knockdown causes a reduction in coilin protein, but not in RNA, levels. VRK1 knockdown effect on coilin results in a disintegration of Cajal bodies. This effect is prevented by proteasome inhibitors. We observed that the loss of coilin phosphorylation permits the ubiquitylation of coilin by Hdm2, but not RNF8, and induces disruption of CBs and, as a consequence, delocalization of SMN1 that we know interacts with both VRK1 and coilin. The effect of VRK1 knockdown on organization can be rescued by VRK1 wt, but not by kinase-dead VRK1 or the R358X mutant. Altered regulation of coilin stability might be implicated in severa! neurological diseases such as and spinal muscular atrophies.

Resumen del trabajo presentado al 5th EMBO Meeting, celebrado en Amsterdam (Holanda) del 21 al 24 de septiembre de 2013.

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