LOV conquers (sm)All GTPases

Knowledge of spatial patterning of GTPases is critical to understanding protein function since subcellular localization is essential for normal protein function. In this paper, we discuss how Hahn’s group has created a new type of genetically encoded, light-activated enzyme that allows precise temporal and spatial control of the small GTPase, Rac1. This reagent enables this group to analyze the roles of effector proteins in Rac1 function and may be applicable to other signaling proteins

Regulation of microfilament reorganization and invasiveness of breast cancer cells by kinase dead p21-activated kinase-1

Stimulation of growth factor signaling has been implicated in the development of invasive phenotype and p21-activated kinase (PAK1) activation in human breast epithelial cancer cells. To further explore the roles of PAK1 in the invasive behavior of breast cancer cells, in the present study we investigated the influence of inhibition of PAK1 activity on the reorganization of cytoskeleton components that control motility and invasiveness of cells, using a highly invasive breast cancer MDA-MB435 as a model system. Our results demonstrate that overexpression of a kinase dead K299R PAK1 mutant leads to suppression of motile phenotypes as well as invasiveness of cells both in the absence or presence of exogenous heregulin-β1. In addition, these phenotypic changes were accompanied by a blockade of disassembly of focal adhesion points, stabilization of stress fibers, and enhanced cell spreading and were dependent on the presence of the kinase dead domain but independent of the presence of the Rac/cdc42 intact (Cdc42/Rac interactive binding) domain of PAK1. We also demonstrated that in K299R PAK1-expressing cells, F-actin filaments were stabilized by persistent co-localization with the actin-binding proteins tropomyosin and caldesmon. Extension of these studies to invasive breast cancer MDA-MB231 cells illustrated that conditional expression of kinase-defective K299R PAK1 was also accompanied by persistent cell spreading, multiple focal adhesion points, and reduced invasiveness. Furthermore, inhibition of PAK1 activity in breast cancer cells was associated with a reduction in c-Jun N-terminal kinase activity, inhibition of DNA binding activity of transcription factor AP-1, and suppression of in vivo transcription driven by AP-1 promoter (known to be involved in breast cancer invasion). These findings suggest that PAK1 downstream pathways have a role in the development and maintenance of invasive phenotypes in breast cancer cells

Targeting and activation of Rac1 are mediated by the exchange factor β-Pix

Rho guanosine triphosphatases (GTPases) are critical regulators of cytoskeletal dynamics and control complex functions such as cell adhesion, spreading, migration, and cell division. It is generally accepted that localized GTPase activation is required for the proper initiation of downstream signaling events, although the molecular mechanisms that control targeting of Rho GTPases are unknown. In this study, we show that the Rho GTPase Rac1, via a proline stretch in its COOH terminus, binds directly to the SH3 domain of the Cdc42/Rac activator β-Pix (p21-activated kinase [Pak]–interacting exchange factor). The interaction with β-Pix is nucleotide independent and is necessary and sufficient for Rac1 recruitment to membrane ruffles and to focal adhesions. In addition, the Rac1–β-Pix interaction is required for Rac1 activation by β-Pix as well as for Rac1-mediated spreading. Finally, using cells deficient for the β-Pix–binding kinase Pak1, we show that Pak1 regulates the Rac1–β-Pix interaction and controls cell spreading and adhesion-induced Rac1 activation. These data provide a model for the intracellular targeting and localized activation of Rac1 through its exchange factor β-Pix

Essential role of CIB1 in regulating PAK1 activation and cell migration

p21-activated kinases (PAKs) regulate many cellular processes, including cytoskeletal rearrangement and cell migration. In this study, we report a direct and specific interaction of PAK1 with a 22-kD Ca2+-binding protein, CIB1, which results in PAK1 activation both in vitro and in vivo. CIB1 binds to PAK1 within discrete regions surrounding the inhibitory switch domain in a calcium-dependent manner, providing a potential mechanism of CIB1-induced PAK1 activation. CIB1 overexpression significantly decreases cell migration on fibronectin as a result of a PAK1-and LIM kinase–dependent increase in cofilin phosphorylation. Conversely, the RNA interference–mediated depletion of CIB1 increases cell migration and reduces normal adhesion-induced PAK1 activation and cofilin phosphorylation. Together, these results demonstrate that endogenous CIB1 is required for regulated adhesion-induced PAK1 activation and preferentially induces a PAK1-dependent pathway that can negatively regulate cell migration. These results point to CIB1 as a key regulator of PAK1 activation and signaling

Interaction with LC8 Is Required for Pak1 Nuclear Import and Is Indispensable for Zebrafish Development

Pak1 (p21 activated kinase 1) is a serine/threonine kinase implicated in regulation of cell motility and survival and in malignant transformation of mammary epithelial cells. In addition, the dynein light chain, LC8, has been described to cooperate with Pak1 in malignant transformation of breast cancer cells. Pak1 itself may aid breast cancer development by phosphorylating nuclear proteins, including estrogen receptor alpha. Recently, we showed that the LC8 binding site on Pak1 is adjacent to the nuclear localization sequence (NLS) required for Pak1 nuclear import. Here, we demonstrate that the LC8-Pak1 interaction is necessary for epidermal growth factor (EGF)-induced nuclear import of Pak1 in MCF-7 cells, and that this event is contingent upon LC8-mediated Pak1 dimerization. In contrast, Pak2, which lacks an LC8 binding site but contains a nuclear localization sequence identical to that in Pak1, remains cytoplasmic upon EGF stimulation of MCF-7 cells. Furthermore, we show that severe developmental defects in zebrafish embryos caused by morpholino injections targeting Pak are partially rescued by co-injection of wild-type human Pak1, but not by co-injection of mutant Pak1 mRNA disrupting either the LC8 binding or the NLS site. Collectively, these results suggest that LC8 facilitates nuclear import of Pak1 and that this function is indispensable during vertebrate development