Jean Paul ten Klooster

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.

Calcium Signaling Regulates Translocation and Activation of Rac

Rac is activated in response to various stimuli including growth factors and by adhesion to the extracellular matrix. However, how these stimuli ultimately result in Rac activation is poorly understood. The increase in intracellular calcium [Ca2+]i represents a ubiquitous second messenger system in cells, linking receptor activation to downstream signaling pathways. Here we show that elevation of [Ca2+]i, either artificially or by thrombin receptor activation, potently induces Rac activation. Lamellipodia formation induced by artificial elevation of [Ca2+]i is blocked by inhibition of Rac signaling, indicating that calcium-induced cytoskeletal changes are controlled by the activation of Rac. Calcium-dependent Rac activation was dependent on the activation of a conventional protein kinase C. Furthermore, both increased [Ca2+]i and protein kinase C activation induce phosphorylation of RhoGDIα and induce the translocation of cytosolic Rac to the plasma membrane. Intracellular calcium signaling may thus contribute to the intracellular localization and activation of Rac to regulate the cytoskeletal changes in response to receptor stimulation.

The C-terminal Domain of Rac1 Contains Two Motifs That Control Targeting and Signaling Specificity

Rho-like GTPases control a wide range of cellular functions such as integrin- and cadherin-mediated adhesion, cell motility, and gene expression. The hypervariable C-terminal domain of these GTPases has been implicated in membrane association and effector binding. We found that cell-permeable peptides, encoding the C termini of Rac1, Rac2, RhoA, and Cdc42, interfere with GTPase signaling in a specific fashion in a variety of cellular models. Pull-down assays showed that the C terminus of Rac1 does not associate to either RhoGDI or to Pak. In contrast, the C terminus of Rac1 (but not Rac2 or Cdc42) binds to phosphatidylinositol 4,5-phosphate kinase (PIP5K) via amino acids 185-187 (RKR). Moreover, Rac1 associates to the adapter protein Crk via the N-terminal Src homology 3 (SH3) domain of Crk and the proline-rich stretch in the Rac1 C terminus. These differential interactions mediate Rac1 localization, as well as Rac1 signaling, toward membrane ruffling, cell-cell adhesion, and migration. These data show that the C-terminal, hypervariable domain of Rac1 encodes two distinct binding motifs for signaling proteins and regulates intracellular targeting and differential signaling in a unique and non-redundant fashion.

Small GTP-Binding Protein Ral Is Involved in cAMP-Mediated Release of von Willebrand Factor From Endothelial Cells

Objective—von Willebrand factor (vWF) is synthesized by endothelial cells and stored in specialized vesicles called Weibel-Palade bodies (WPBs). Recently, we have shown that the small GTP-binding protein Ral is involved in thrombin-induced exocytosis of WPBs. In addition to Ca2+-elevating secretagogues such as histamine and thrombin, release of WPB is also observed after administration of cAMP-raising substances such as epinephrine and vasopressin. In the present study, we investigated whether Ral is also involved in cAMP-mediated vWF release. Methods and Results—Activation of Ral was observed 15 to 20 minutes after stimulation of endothelial cells with epinephrine, forskolin, or dibutyryl-cAMP. A cell-permeable peptide comprising the carboxy-terminal part of the Ral protein reduced both thrombin-induced and epinephrine-induced vWF secretion supporting a crucial role for Ral in this process. Furthermore, inhibition of protein kinase A by H-89 resulted in a marked reduction of vWF release and greatly diminished levels of GTP-Ral on stimulation with epinephrine. Activation of Ral was independent of the activation of Epac, a cAMP-regulated exchange factor for the small GTPases Rap1 and Rap2. Conclusions—These results suggest that protein kinase A-dependent activation of Ral regulates cAMP-mediated exocytosis of WPB in endothelial cells.