Differential Inhibitor Sensitivity between Human Kinases VRK1 and VRK2

Human vaccinia-related kinases (VRK1 and VRK2) are atypical active Ser-Thr kinases implicated in control of cell cycle entry, apoptosis and autophagy, and affect signalling by mitogen activated protein kinases (MAPK). The specific structural differences in VRK catalytic sites make them suitable candidates for development of specific inhibitors. In this work we have determined the sensitivity of VRK1 and VRK2 to kinase inhibitors, currently used in biological assays or in preclinical studies, in order to discriminate between the two proteins as well as with respect to the vaccinia virus B1R kinase. Both VRK proteins and vaccinia B1R are poorly inhibited by inhibitors of different types targeting Src, MEK1, B-Raf, JNK, p38, CK1, ATM, CHK1/2 and DNA-PK, and most of them have no effect even at 100 µM. Despite their low sensitivity, some of these inhibitors in the low micromolar range are able to discriminate between VRK1, VRK2 and B1R. VRK1 is more sensitive to staurosporine, RO-31-8220 and TDZD8. VRK2 is more sensitive to roscovitine, RO 31–8220, Cdk1 inhibitor, AZD7762, and IC261. Vaccinia virus B1R is more sensitive to staurosporine, KU55933, and RO 31–8220, but not to IC261. Thus, the three kinases present a different pattern of sensitivity to kinase inhibitors. This differential response to known inhibitors can provide a structural framework for VRK1 or VRK2 specific inhibitors with low or no cross-inhibition. The development of highly specific VRK1 inhibitors might be of potential clinical use in those cancers where these kinases identify a clinical subtype with a poorer prognosis, as is the case of VRK1 in breast cancer

Efecto de diferentes inhibidores y metales sobre las quinasas humanas VRK y la proteína viral B1R

Memoria presentada por Iria Barcia Sanjurjo, graduada en Ciencia y Tecnología de los Alimentos para optar al grado de Doctor en Bioquímica y Biología Molecular por la Universidad de Santiago de Compostela (Departamento de Bioquímica y Biología Molecular) y realizada en el Instituto de Biología Molecular y Celular del Cancer de Salamanca.Esta Tesis Doctoral ha sido realizada dentro del Proyecto de Investigación Zimofeed: 08MMA003CT financiado por la Secretaria Xeral de Investigación e Desenvolvemento da Xunta de Galicia. Doña Iria Barcia Sanjurjo ha disfrutado de un contrato predoctoral financiado por la Vicerrectoría de Investigación e Innovación junto con el Departamento de Bioquímica y Biología Molecular de la Universidad de Santiago de Compostela. La investigación en el laboratorio del Centro de Investigación del Cancer (CIC-C.S.I.C) ha sido financiada por los siguientes proyectos: Ministerio de Educación y Ciencia (SAF2004-02900; SAF2007-60242; CDS-2007-0017 y SAF2010-14935). Junta de Castilla y León, Consejería de Sanidad (SAN/673/SA05/08; BOCyL Nº 170). Junta de Castilla y León, Consejería de Educación (CSI14A08; CSI006A11-2 y grupo de excelencia GR-15). Kutxa-Fundación INBIOMED.Peer reviewe

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

The human vaccinia-related kinase (VRK) proteins VRK1 and VRK2 regulate different processes, such as the cell cycle, DNA damage response, and signaling by mitogen-activated protein kinases in response to growth factors or cellular stress. Alterations in expression levels of these Ser-Thr kinases are associated with cancer and neurodegenerative diseases. These functions suggest that they might also be targets of toxic metals, and thus contribute to the pathogenic effects associated with metal intoxication. VRK1 is inhibited by cadmium, copper, and mercury, and VRK2 is more sensitive to cadmium and much less sensitive to copper and mercury. Both kinases are insensitive to lead and cobalt. VRK1 is in general more sensitive than VRK2 in the low micromolar range. This inhibitory effect induced by these metals was detected in an autophosphorylation assay, as well as in phosphorylation assays using p53 and histone H3 as substrates. The accumulation of these three metals in cells can contribute, by inhibition of VRKs, to their toxic pathogenic effects, particularly their neurological manifestations. In this context copper has not generally been associated with any intoxication syndrome, except Wilson's syndrome, but it might be implicated in some alterations with which it has not yet been associated.I.B.-S. and M.V.-C. have fellowships from Universidad de Santiago and JAE/CSIC/Fondo Social Europeo respectively. This work was funded by grants from Ministerio de Educación, Ciencia e Innovación (SAF2010-14935), Junta de Castilla y León (CSI-006A11-2), and Kutxa-Fundación INBIOMED to P.A.L, and from Xunta de Galicia to R. B.Peer Reviewe