Substrate profiling of human vaccinia-related kinases identifies coilin, a Cajal body nuclear protein, as a phosphorylation target with neurological implications

Abstract

Protein phosphorylation by kinases plays a central role in the regulation and coordination of multiple biological processes. In general, knowledge on kinase specificity is restricted to substrates identified in the context of specific cellular responses, but kinases are likely to have multiple additional substrates and be integrated in signaling networks that might be spatially and temporally different, and in which protein complexes and subcellular localization can play an important role. In this report the substrate specificity of atypical human vaccinia-related kinases (VRK1 and VRK2) using a human peptide-array containing 1080 sequences phosphorylated in known signaling pathways has been studied. The two kinases identify a subset of potential peptide targets, all of them result in a consensus sequence composed of at least four basic residues in peptide targets. Linear peptide arrays are therefore a useful approach in the characterization of kinases and substrate identification, which can contribute to delineate the signaling network in which VRK proteins participate. One of these target proteins is coilin; a basic protein located in nuclear Cajal bodies. Coilin is phosphorylated in Ser184 by both VRK1 and VRK2. Coilin colocalizes and interacts with VRK1 in Cajal bodies, but not with the mutant VRK1 (R358X). VRK1 (R358X) is less active than VRK1. Altered regulation of coilin might be implicated in several neurological diseases such as ataxias and spinal muscular atrophies.

VRK1 and AURKB form a complex that cross inhibit their kinase activity and the phosphorylation of histone H3 in the progression of mitosis

“…The expression and purification of glutathione S -transferase (GST)-tagged proteins was performed with the resin Glutathione Sepharose 4B as previously reported [6, 27, 28, 45, 70]. The expression and purification of the protein was performed using the E. coli Bl21DE3 strain that was transformed with a pGEX–4T–GST plasmid containing the protein to express (pGEX4T–VRK1).…”

“…The Ser-Thr kinase activity of either VRK1 or AURKB was performed under the same experimental conditions through in vitro kinase assays using radiolabeled ATP [γ- 32 P] [6, 27, 47]. This technique was performed using either GST-fusion kinase proteins expressed in E. coli or immunoprecipitated (endogenous or transfected) protein kinase from cell extracts.…”

“…VRK1 is mostly a chromatin kinase and regulates histones [9, 26], but is also present in other complexes of nucleic acids, including RNA, and nucleoproteins, as exemplified by the complex with coilin [27, 28], the scaffold protein of Cajal bodies [29]. It is possible that VRK1 plays complementary roles, as a key regulator of chromatin dynamics in different situations ranging from relaxation [26] to compaction [9] at the entry of mitosis.…”

Sensitivity of the kinase activity of human vaccinia-related kinase proteins to toxic metals

“…The elimination of VRK1 in gene-trap mice has revealed that reduced levels of this kinase play an important role in meiosis, resulting in male and female sterility [47][48][49]. Also, the loss in mice of coilin, a phosphorylation target of VRK1 [50], is manifested by a sterility phenotype [51]. Thus, long-term accumulation of cadmium or mercury could probably result in reduced fertility in intoxicated individuals.…”

Specific genomic cues regulate Cajal body assembly

“…To date, 18 distinct human coilin phosphorylation residues have been reported, with several known regulators of their modification, including the phosphatase PPM1G. 106 Cell cycledependent phosphorylation, potentially by the Serine-Threonine kinase VRK1, 55,107 has also been linked to structural integrity of the CB. Differential coilin phosphorylation may mislocalize CBs to the intra-nucleolar space, potentially resulting in aberrant snRNP processing.…”