Abstract
MRCKα and MRCKβ (myotonic dystrophy kinase-related Cdc42-binding kinases) belong to a subfamily of Rho GTPase activated serine/threonine kinases within the AGC-family that regulate the actomyosin cytoskeleton. Reflecting their roles in myosin light chain (MLC) phosphorylation, MRCKα and MRCKβ influence cell shape and motility. We report further evidence for MRCKα and MRCKβ contributions to the invasion of cancer cells in 3-dimensional matrix invasion assays. In particular, our results indicate that the combined inhibition of MRCKα and MRCKβ together with inhibition of ROCK kinases results in significantly greater effects on reducing cancer cell invasion than blocking either MRCK or ROCK kinases alone. To probe the kinase ligand pocket, we screened 159 kinase inhibitors in an in vitro MRCKβ kinase assay and found 11 compounds that inhibited enzyme activity >80% at 3 µM. Further analysis of three hits, Y-27632, Fasudil and TPCA-1, revealed low micromolar IC50 values for MRCKα and MRCKβ. We also describe the crystal structure of MRCKβ in complex with inhibitors Fasudil and TPCA-1 bound to the active site of the kinase. These high-resolution structures reveal a highly conserved AGC kinase fold in a typical dimeric arrangement. The kinase domain is in an active conformation with a fully-ordered and correctly positioned αC helix and catalytic residues in a conformation competent for catalysis. Together, these results provide further validation for MRCK involvement in regulation of cancer cell invasion and present a valuable starting point for future structure-based drug discovery efforts.
Highlights
Tumor cell metastasis is a multi-step process driven by dynamic reorganization of the actomyosin cytoskeleton and remodeling of the extracellular matrix that allows cells to cross tissue boundaries and spread via blood and lymphatic vessels to distal regions of the body [1]
Given the potential for off-target effects of Y-27632, on highly homologous kinases such as MRCK, we knocked down ROCK 1 and/or ROCK2 to corroborate the effects of ROCK inhibition (Figure 1D)
Previous studies have shown that the combination of MRCK as well as ROCK inhibition has greater effects in blocking the invasiveness of tumor cells than inhibition of either kinase alone [17]
Summary
Tumor cell metastasis is a multi-step process driven by dynamic reorganization of the actomyosin cytoskeleton and remodeling of the extracellular matrix that allows cells to cross tissue boundaries and spread via blood and lymphatic vessels to distal regions of the body [1]. The bundling and contraction of actin-myosin fibers provides the force required for cell motility and invasion [1] On this basis, downstream effector proteins such as the Rho-regulated ROCK1 and ROCK2 protein kinases that directly impact upon actomyosin contractility have emerged as attractive potential targets for anti-metastatic therapeutics [3,4]. Cells that migrate through 3-dimensional (3-D) extracellular matrix (ECM) with a rounded morphology ( known as amoeboid invasion) are more dependent upon ROCK activity, whereas cells that invade using elongated actin-rich protrusions ( called mesenchymal invasion) are relatively insensitive to ROCK inhibition [15,16,17,18] Both invasion modes are dependent upon the contractile force generated by myosin ATPase activity [17], indicating that regulators of actomyosin function in addition to ROCK are involved
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
