Rho Kinase Differentially Regulates Phosphorylation of Nonmuscle Myosin II Isoforms A and B during Cell Rounding and Migration

The actin-myosin cytoskeleton is generally accepted to produce the contractile forces necessary for cellular processes such as cell rounding and migration. All vertebrates examined to date are known to express at least two isoforms of non-muscle myosin II, referred to as myosin IIA and myosin IIB. Studies of myosin IIA and IIB in cultured cells and null mice suggest that these isoforms perform distinct functions. However, how each myosin II isoform contributes individually to all the cellular functions attributed to "myosin II" has yet to be fully characterized. Using isoform-specific small-interfering RNAs, we found that depletion of either isoform resulted in opposing migration phenotypes, with myosin IIA- and IIB-depleted cells exhibiting higher and lower wound healing migration rates, respectively. In addition, myosin IIA-depleted cells demonstrated impaired thrombin-induced cell rounding and undertook a more motile morphology, exhibiting decreased amounts of stress fibers and focal adhesions, with concomitant increases in cellular protrusions. Cells depleted of myosin IIB, however, were efficient in thrombin-induced cell rounding, displayed a more retractile phenotype, and maintained focal adhesions but only in the periphery. Last, we present evidence that Rho kinase preferentially regulates phosphorylation of the regulatory light chain associated with myosin IIA. Our data suggest that the myosin IIA and IIB isoforms are regulated by different signaling pathways to perform distinct cellular activities and that myosin IIA is preferentially required for Rho-mediated contractile functions

MLK3 Limits Activated Gαq Signaling to Rho by Binding to p63RhoGEF

Mixed lineage kinase 3 (MLK3) is a MAP3K that activates the JNK-dependent MAPK pathways. Here we show that MLK3 is required for cell migration in a manner independent of its role as a MAP3K or MLK3 kinase activity. Rather, MLK3 functions in a regulated way to limit levels of the activated GTPase, Rho, by binding to the Rho activator, p63RhoGEF/GEFT, which, in turn, prevents its activation by Gαq. These findings demonstrate a scaffolding role for MLK3 in controlling the extent of Rho activation that modulates cell migration. Moreover, they suggest that MLK3 functions as a network hub that links a number of signaling pathways

Distinct Functions and Regulation of Nonmuscle Myosin II Isoforms a and B in Cell Motility

<p>The ability of cells to migrate is of fundamental importance to a diverse array of biological processes, both physiological and pathological, such as development, the immune response and cancer cell metastasis, to name a few. The process of cell movement is a complicated cycle of coordinated steps involving dynamic and precise rearrangement of the actin-myosin cytoskeleton. As a critical component of the migration machinery, the molecular motor protein nonmuscle myosin II (myosin II) has long been a subject of scientific inquiry. It is now generally accepted that the contractile forces generated by myosin II contribute directly or indirectly to every step in migration. Interestingly, three isoforms of myosin II (myosin IIA, IIB and IIC) have been identified, and although each isoform performs the same basic molecular functions, recent findings suggest that the different myosin II isoforms make unique contributions to the motile process. In this dissertation work I used RNA interference technology to specifically deplete cells of myosin IIA and IIB in order to characterize the distinct migration phenotypes associated with loss-of-function of each individual isoform. Surprisingly, I found that the two myosin II isoforms perform not only distinct but opposing functions in cell migration, with myosin IIA and IIB normally inhibiting and facilitating proper cell movement, respectively. Furthermore, using pharmacological and microscopy techniques, I investigated the cellular mechanisms allowing for isoform-specific function. My results provide evidence for at least two isoform-specific regulatory mechanisms, namely selectivity in signaling pathways and subcellular distribution. A particularly significant finding is the identification of the different assembly properties of myosin IIA and IIB as the key element responsible for directing isoform-distinct distribution. Together the data presented herein represent a considerable advance in our understanding of the distinct functions and regulation of myosin II in cell motility.</p>Dissertatio

The C-Terminal Tail Region of Nonmuscle Myosin II Directs Isoform-specific Distribution in Migrating Cells

Nonmuscle myosin II isoforms A and B (hereafter, IIA and IIB) perform unique roles in cell migration, even though both isoforms share the same basic molecular functions. That IIA and IIB assume distinct subcellular distribution in migrating cells suggests that discrete spatiotemporal regulation of each isoform's activity may provide a basis for its unique migratory functions. Here, we make the surprising finding that swapping a small C-terminal portion of the tail between IIA and IIB inverts the distinct distribution of these isoforms in migrating cells. Moreover, swapping this region between isoforms also inverts their specific turnover properties, as assessed by fluorescence recovery after photobleaching and Triton solubility. These data, acquired through the use of chimeras of IIA and IIB, suggest that the C-terminal region of the myosin heavy chain supersedes the distinct motor properties of the two isoforms as the predominant factor directing isoform-specific distribution. Furthermore, our results reveal a correlation between isoform solubility and distribution, leading to the proposal that the C-terminal region regulates isoform distribution by tightly controlling the amount of each isoform that is soluble and therefore available for redistribution into new protrusions