ROCK Cellular Phosphorylation Assay Service

The Rho-associated, coiled-coil containing serine/threonine kinases ROCK I and ROCK II (ROCK) are involved in the regulation of cytoskeletal activity. Activation of ROCK by binding to activated Rho-GTP results in the phosphorylation of numerous substrate proteins like regulatory myosin light chain (MLC) or the myosin binding subunit of MLC phosphatase (MYPT1). These phosphorylations lead to changes in cell morphology causing phenotypes like cellular contraction, formation of stress fibers or neurite retraction. Several of these processes play important roles in pathological conditions like cardiovascular diseases, neuronal degeneration or cancer.

ROCK1 and ROCK2

P160ROCK, KIAA0619, ROC2

A7r5

Endogenous

The myosin light chain (MLC) in the rat aortic smooth muscle cell line A7r5 is constitutively bisphosphorylated at Thr18/Ser19 by ROCK kinases, which directly phosphorylate MLC and simultaneously prevent its dephosphorylation through inhibition of MLC phosphatase. Inhibition of ROCK kinase by specific inhibitors like Y27632 reduce the levels of phospho-MLC (see Fig.1). In the cellular assay phospho-levels of MLC-Thr18/Ser19 are quantified by direct-ELISA technique.

Substrate phosphorylation as a readout of intracellular kinase activity via ELISA

Freiburg, Germany

More information can be found on our website Cellular Phosphorylation Assay Services.