ROCKII Ser1366 phosphorylation reflects the activation status

Abstract

ROCK (Rho-associated protein kinase), a downstream effector of RhoA, plays an important role in many cellular processes. Accumulating evidence has shown the involvement of ROCK activation in the pathogenesis of many diseases. However, a reagent capable of detecting ROCK activation directly is lacking. In the present study, we show autophosphorylation of ROCKII in an in vitro kinase reaction. The phosphorylation sites were identified by MS, and the major phosphorylation site was found to be at the highly conserved residue Ser1366. A phospho-specific antibody was generated that can specifically recognize ROCKII Ser1366 phosphorylation. We found that the extent of Ser1366 phosphorylation of endogenous ROCKII is correlated with that of myosin light chain phosphorylation in cells in response to RhoA stimulation, showing that Ser1366 phosphorylation reflects its kinase activity. In addition, ROCKII Ser1366 phosphorylation could be detected in human breast tumours by immunohistochemical staining. The present study provides a new approach for revealing the ROCKII activation status by probing ROCKII Ser1366 phosphorylation directly in cells or tissues.

A novel small-molecule MRCK inhibitor blocks cancer cell invasion

“…In addition, these reagents would have the potential to be useful as pharmacodynamic markers of in vivo drug effects. This approach has been used for ROCK1 and ROCK2, where autophosphorylation on Ser1333 and Ser1366 respectively were determined to reflect activity [27,28]. In the case of ROCK2, phosphoselective antibodies against this Ser1366 post-translational modification were used to stain breast cancer clinical samples to detect ROCK2 activation.…”

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“…4b Costa et al 2013). In the LPP terminal field, WIN increased levels of pROCK S1366 (Chuang et al 2012) co-localized with SYN (P < 0.0001; Fig. 4c) but not with the postsynaptic density marker PSD-95 (P = 0.74; Fig.…”

“…The microscope slide mounted tissue was fixed in 4% paraformaldehyde and processed for immunostaining as described (Seese et al 2014). Immunofluorescence procedures used cocktails of primary antisera (Table 1) including rabbit antisera to pMunc18-1 Ser241 (Schmitz et al 2016), phosphorylated proline-rich tyrosine kinase 2 (pPyk2) Tyr402 (Zhao et al 2000), phosphorylated RhoA associated coiled coil containing kinase 2 (pROCK2) Ser1366 (Chuang et al 2012), or pFAK Y397 (Bock and Herz 2003), used in combination with mouse antisera to synaptophysin (SYN) (Wen et al 2014) or postsynaptic density protein-95 (PSD-95) (Takahashi et al 2011); or guinea pig anti-vGluT2 used in combination with rabbit anti-CB 1 (Matyas et al 2006) that was generously provided by Dr Ken Mackie of Indiana University. Secondary antisera from ThermoFisher Scientific included Alexa Fluor 594 antirabbit IgG (A21207), Alexa Fluor 488 anti-mouse IgG (A21202) and Alexa Fluor 488 anti-guinea pig IgG (A-11073) all used at 1:1000 dilutions.…”

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“…Finally, the identification and development of MRCK selective pharmacodynamics biomarkers would be useful for monitoring drug action, and possibly for identifying tumor types with evidence of significant MRCK activity. Autophosphorylation of ROCK2 on Serine 1366 was shown to reflect its activity status in cells, while a phosphorylation-sensitive antibody reagent revealed increased ROCK2 activity in breast cancer (19). Given the high homology between MRCK and ROCK kinases, a similar approach could be used to identify MRCK autophosphorylation events that were indicative of kinase activity and which could be recognized by phosphorylation-sensitive antibodies for pharmacodynamics studies.…”