Abstract
Human vaccinia-related kinase (VRK) 1 is a novel serine-threonine kinase that regulates several transcription factors, nuclear envelope assembly, and chromatin condensation and is also required for cell cycle progression. The regulation of this kinase family is unknown. Mass spectrometry has permitted the identification of Ran as an interacting and regulatory protein of the VRK serine-threonine kinase activities. The stable interaction has been validated by pulldown of endogenous proteins as well as by reciprocal immunoprecipitations. The three members of the VRK family stably interact with Ran, and the interaction was not affected by the bound nucleotide, GDP or GTP. The interaction was stronger with the RanT24N that is locked in its inactive conformation and cannot bind nucleotides. None of the kinases phosphorylated Ran or RCC1. VRK1 does not directly interact with RCC1, but if Ran is present they can be isolated as a complex. The main effect of the interaction of inactive RanGDP with VRK1 is the inhibition of its kinase activity, which was detected by a reduction in VRK1 autophosphorylation and a reduction in phosphorylation of histone H3 in residues Thr-3 and Ser-10. The kinase activity inhibition can be relieved by the interaction with the constitutively active RanGTP or RanL43E, which locks Ran in its GTP-bound active conformation. In this complex, the interaction with VRK proteins does not alter the effect of its guanine exchange factor, RCC1. Ran is a novel negative regulator of nuclear VRK1 and VRK2 kinase activity, which may vary in different subcellular localizations generating an asymmetric intracellular distribution of kinase activity depending on local protein interactions.
Highlights
Human vaccinia-related kinase (VRK) 1 is a novel serinethreonine kinase that regulates several transcription factors, nuclear envelope assembly, and chromatin condensation and is required for cell cycle progression
Cell extracts were used for a pulldown of associated proteins using as control GST versus GST-VRK2B or GSTVRK1
SYPRO Ruby staining allowed the detection of several different spots pulled down with GST-VRK2B or GST-VRK1 but not with control GST that may correspond to proteins bound to VRK proteins
Summary
Plasmids—Plasmid pCEFL-GST expresses GST fusion protein in mammalian cells. VRK1 full-length cDNA was subcloned in vector pCEFL-GST with restriction sites BamHI-BamHI. GST-VRK proteins or immunoprecipitated VRK1 was incubated previously with GST-Ran purified proteins or with buffer only 1 h at 4 °C to allow the interaction before starting the phosphorylation reactions. GST-Ran (1 nmol) in loading buffer (20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.1% (v/v) Triton X-100, 1 mM MgCl2, and 6 mM EDTA) was incubated with 0.185 MBq of [3H]GDP (5 Ci) (GE Healthcare TRK 335) in a final volume of 50 l for 30 min at 24 °C. Reaction mixtures contained RCC1 and VRK1 in exchange buffer (20 mM Tris-HCl, pH 7.5, 100 mM NaCl, and 0.1% Triton X-100) to give final protein concentrations indicated in the figures and free unlabeled GTP for exchange in a final concentration of 2 mM. The incorporated radioactivity was measured at the indicated times in the figures
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