Biomedical subjects

Ritu Garg

Publications and source records attributed to Ritu Garg.

6 recordsLinked to original sources

PubMed2006

Mammalian cell microinjection assay to study the function of Rho family guanosine triphosphatases.

Microinjection is an excellent technique for studying the acute responses of cells to proteins and can be used to investigate the effects of mutations in proteins on their activity. It has been used widely to study the responses to Rho family guanosine triphosphatases and is particularly useful for cell types that are difficult to transfect. Here, we describe the procedure for microinjecting cells with purified recombinant proteins or with expression vectors encoding proteins, and for analyzing the cells after injection.

3T3 Cells↗

PubMed2005

RhoE function is regulated by ROCK I-mediated phosphorylation.

The Rho GTPase family member RhoE regulates actin filaments partly by binding to and inhibiting ROCK I, a serine/threonine kinase that induces actomyosin contractility. Here, we show that ROCK I can phosphorylate multiple residues on RhoE in vitro. In cells, ROCK I-phosphorylated RhoE localizes in the cytosol, whereas unphosphorylated RhoE is primarily associated with membranes. Phosphorylation has no effect on RhoE binding to ROCK I, but instead increases RhoE protein stability. Using phospho-specific antibodies, we show that ROCK phosphorylates endogenous RhoE at serine 11 upon cell stimulation with platelet-derived growth factor, and that this phosphorylation requires an active protein kinase C signalling pathway. In addition, we demonstrate that phosphorylation of RhoE correlates with its activity in inducing stress fibre disruption and inhibiting Ras-induced transformation. This is the first demonstration of an endogenous Rho family member being phosphorylated in vivo and indicates that phosphorylation is an important mechanism to control the stability and function of this GTPase-deficient Rho protein.

Animals↗

PubMed2003

The prostate-derived sterile 20-like kinase (PSK) regulates microtubule organization and stability.

Sterile 20 (STE20) protein kinases, which include germinal center kinases and p21-activated protein kinases, are known to activate mitogen-activated protein kinase pathways (c-Jun NH(2)-terminal kinase, p38, or extracellular signal-regulated kinase), leading to changes in gene transcription. Some STE20s can also regulate the cytoskeleton, and we have shown that the germinal center kinase-like kinase prostate-derived STE20-like kinase (PSK) affects actin cytoskeletal organization. Here, we demonstrate that PSK colocalizes with microtubules; and that this localization is disrupted by the microtubule depolymerizing agent nocodazole. The association of PSK with microtubules results in the production of stabilized perinuclear microtubule cables that are nocodazole-resistant and contain increased levels of acetylated alpha-tubulin. Kinase-defective PSK (K57A) or the C terminus of PSK (amino acids 745-1235) lacking the kinase domain are sufficient for microtubule binding and stabilization, demonstrating that the catalytic activity of the protein is not required. The localization of PSK to microtubules occurs via its C terminus, and PSK binds and phosphorylates alpha- and beta-tubulin in vitro. The N terminus of PSK (1-940) is unable to bind or stabilize microtubules, demonstrating that PSK must associate with microtubules for their reorganization to occur. These results demonstrate that PSK interacts with microtubules and affects their organization and stability independently of PSK kinase activity.

3T3 Cells↗

PubMed2003

RhoE binds to ROCK I and inhibits downstream signaling.

RhoE belongs to the Rho GTPase family, the members of which control actin cytoskeletal dynamics. RhoE induces stress fiber disassembly in a variety of cell types, whereas RhoA stimulates stress fiber assembly. The similarity of RhoE and RhoA sequences suggested that RhoE might compete with RhoA for interaction with its targets. Here, we show that RhoE binds ROCK I but none of the other RhoA targets tested. The interaction of RhoE with ROCK I was confirmed by coimmunoprecipitation of the endogenous proteins, and the two proteins colocalized on the trans-Golgi network in COS-7 cells. Although RhoE and RhoA were not able to bind ROCK I simultaneously, RhoE bound to the amino-terminal region of ROCK I encompassing the kinase domain, at a site distant from the carboxy-terminal RhoA-binding site. Overexpression of RhoE inhibited ROCK I-induced stress fiber formation and phosphorylation of the ROCK I target myosin light chain phosphatase. These data suggest that RhoE induces stress fiber disassembly by directly binding ROCK I and inhibiting it from phosphorylating downstream targets.

3T3 Cells↗

PubMed2002

Phosphorylation of tyrosine 291 enhances the ability of WASp to stimulate actin polymerization and filopodium formation. Wiskott-Aldrich Syndrome protein.

Wiskott-Aldrich Syndrome protein (WASp) is a key regulator of the Arp2/3 complex and the actin cytoskeleton in hematopoietic cells. WASp is capable of forming an auto-inhibited conformation, which can be disrupted by binding of Cdc42 and phosphatidylinositol 4,5-bisphosphate, leading to its activation. Stimulation of the collagen receptor on platelets and crosslinking the B-cell receptor induce tyrosine phosphorylation of WASp. Here we show that the Src family kinase Hck induces phosphorylation of WASp-Tyr(291) independently of Cdc42 and that this causes a shift in the mobility of WASp upon SDS-PAGE. A phospho-mimicking mutant, WASp-Y291E, exhibited an enhanced ability to stimulate actin polymerization in a cell-free system and when microinjected into primary macrophages induced extensive filopodium formation with greater efficiency than wild-type WASp or a Y291F mutant. We propose that phosphorylation of Tyr(291) directly regulates WASp function.

Actins↗

PubMed2002

Extension, retraction and contraction in the formation of a dendritic cell dendrite: distinct roles for Rho GTPases.

The morphology of antigen-presenting dendritic cells (DC) is characterized by the possession of numerous long arborizing processes known as dendrites. The formation of these processes by DC, both in the periphery and in lymphoid organs, is believed to contribute to the remarkable efficiency with which they take up, process and present antigen to T cells. However, the process of dendrite formation and the signaling pathways that lead to the formation of these dendrites remain obscure. In this study we describe an in vitro model in which human immature DC form long processes similar to those formed in vivo. The formation of these processes involves initial attachment of a cell protrusion to the extracellular matrix substrate, and subsequent movement of the cell body away from the adhesion site, leaving behind a long slender dendrite. Dendrite formation, but not their maintenance, was found to be dependent on the activity of Rho GTPases. More specifically, Cdc42 and Rac1 were both required for the migration step of process formation, promoting cell spreading and extension. In contrast, Rho, and its downstream effector p160ROCK, regulated the release of adhesions to the substratum, and associated cellular contraction. Consequently, inhibition of Rho/p160ROCK leads to the formation of longer dendrites. DC therefore coordinate adhesion and protrusion to perform a specialized process of cellular morphogenesis, which differentiates these cells from all other cells of the immune system and may contribute to their distinctive function.

Cell Adhesion↗