Differential Inhibitor Sensitivity between Human Kinases VRK1 and VRK2

Abstract

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.

Vaccinia-related Kinase 1 (VRK1) Is an Upstream Nucleosomal Kinase Required for the Assembly of 53BP1 Foci in Response to Ionizing Radiation-induced DNA Damage

“…1B). The activity of VRK1 is known to be insensitive to caffeine (17), an inhibitor of PI3K-related kinases, such as ATM and ATR, and to KU55933, an ATM-specific inhibitor (44). The arrested status of the cell cycle was confirmed by a reduction in phospho-Rb and cyclin D1 and an increase in p27 inhibitor levels (Fig.…”

VRK1 chromatin kinase phosphorylates H2AX and is required for foci formation induced by DNA damage

“…54 Alternatively, high VRK1 levels should make cells more resistant to treatment, and this has been reported in breast cancer, 54 in which VRK1 identifies a subgroup within estrogen receptor positive cases that presents a poorer prognosis. 64,65 The structure of VRK1 indicates that any specific inhibitor will have no crossinhibitory effect on other kinases due its very low promiscuity index 55,56 and, therefore, makes VRK1 a very good candidate for development of highly specific inhibitors that may be useful in treatment of some tumors.…”

“…VRK1 is insensitive to known kinase inhibitors targeting all kinases families in the kinome 55 and has a very low promiscuity (cross inhibitory) index, 56 making it a potential target for highly specific inhibitors. Several kinases participating in DDR are members of the PI3K family.…”

“…Several kinases participating in DDR are members of the PI3K family. Inhibitors of PI3K such as caffeine, 57 which does not inhibit VRK1, 35 and KU55933, which specifically inhibits ATM 58 but not VRK1, 55 were tested on gH2AX foci induced by IR. Each inhibitor by itself was able to reduce formation of gH2AX foci induced by IR, consistent with the known role of ATM in DDR signaling.…”

Human VRK2 (Vaccinia-related Kinase 2) Modulates Tumor Cell Invasion by Hyperactivation of NFAT1 and Expression of Cyclooxygenase-2

“…Immunoprecipitates were washed and used for an in vitro kinase assay with 1 g of GST-VRK2 in a final volume of 40 l containing kinase buffer (20 mM Tris-HCl, pH 7.5, 5 mM MgCl 2 , 0,5 mM DTT, and 150 mM KCl), 5 M ATP, and 5 Ci of [␥-32 P]ATP. Purified GST-NFAT constructs and GST-VRK proteins were used for in vitro kinase assays in a final volume of 30 l in the same conditions described previously (24,55). The reactions were performed at 30°C for 30 min and stopped by boiling in Laemmli buffer.…”