View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector A Rac-cGMP Signaling Pathway Dagang Guo,1 Ying-cai Tan,1 Dawei Wang,1 K.S. Madhusoodanan,1 Yi Zheng,2 Thomas Maack,1 J. Jillian Zhang,1 and Xin-Yun Huang1,* 1 Department of Physiology, Cornell University Weill Medical College, New York, NY 10021, USA 2 Division of Experimental Hematology, Children’s Hospital Research Foundation, University of Cincinnati, Cincinnati, OH 45229, USA *Correspondence: [email protected] DOI 10.1016/j.cell.2006.11.048 SUMMARY et al., 2004). PAK family members regulate cellular prolif- eration, differentiation, transformation, and survival. They The small GTPase Rac and the second messen- also play important roles in cytoskeleton rearrangement ger cGMP (guanosine 30,50-cyclic monophos- during cell migration. Expression of constitutively active phate) are critical regulators of diverse cell PAK stimulates ruffle formation and inhibits stress fibers functions. When activated by extracellular (Manser et al., 1997; Sells et al., 1997). Increases in PAK signals via membrane signaling receptors, Rac expression and activity have been correlated with pro- executes its functions through engaging down- gression of colorectal carcinomas to metastasis (Carter et al., 2004) and enhanced motility and invasiveness of stream effectors such as p21-activated kinase human breast cancer cells (Vadlamudi et al., 2000). In (PAK), a serine/threonine protein kinase. How- mammals, PAKs can be grouped into two subfamilies: ever, the molecular mechanism by which mem- group A (PAK1, 2, and 3) can be activated by small brane signaling receptors regulate cGMP levels GTPases such as Rac-GTP or Cdc42-GTP binding is not known. Here we have uncovered a sig- (Bokoch, 2003). Group B (PAK4, 5, and 6) can interact naling pathway linking Rac to the increase with Cdc42-GTP but are not activated by this binding. of cellular cGMP. We show that Rac uses PAK From the crystal structure of PAK1 (Lei et al., 2000), it to directly activate transmembrane guanylyl appears that the inactive state is maintained by the N- cyclases (GCs), leading to increased cellular terminal autoregulatory region (residues 83–149) binding cGMP levels. This Rac/PAK/GC/cGMP path- to and inhibiting the C-terminal catalytic kinase domain. way is involved in platelet-derived growth This autoregulatory region inhibits PAK1 even when sup- plied as an independent fragment. Binding of GTP bound factor-induced fibroblast cell migration and Rac (or active Cdc42) releases this inhibition. PAK1 then lamellipodium formation. Our findings connect autophosphorylates Thr423 in its activation loop to stabi- two important regulators of cellular physiologi- lize the active state (Lei et al., 2000). In addition, PAK1, cal functions and provide a general mechanism 2, and 3 can be activated by Rac/Cdc42-independent for diverse receptors to modulate physiological mechanisms such as by caspase-mediated cleavage, by responses through elevating cellular cGMP membrane recruitment via adaptor proteins, and by levels. sphingolipids (Bokoch, 2003). Like cAMP and Ca2+, cGMP is a ubiquitous second messenger mediating cellular responses to various exog- INTRODUCTION enous and endogenous signaling molecules. cGMP con- trols diverse physiological functions such as relaxation The small GTPase Rac plays critical roles in a wide variety of vascular smooth muscles, phototransduction, epithelial of cell functions, including cell-cycle control, regulation of electrolyte transport, bone growth, leukocyte migration, gene expression, activation of NADPH oxidase of phago- axonal guidance, sperm motility, platelet spreading, and cytic cells, actin cytoskeletal reorganization, axonal guid- vascular permeability (Lucas et al., 2000). cGMP regulates ance, and cell migration (Jaffe and Hall, 2005). Rac can be physiological processes by activating protein kinases, activated by extracellular signals through various types of gating specific ion channels and modulating cellular cyclic membrane receptors, including receptor tyrosine kinases nucleotide concentrations through phosphodiesterases such as the PDGF (platelet-derived growth factor) recep- (Lucas et al., 2000). The conversion of GTP to cGMP is tor. Rac executes its biological functions through activat- catalyzed by guanylyl cyclases (GCs). There are two types ing downstream effectors. One of the best-characterized of GCs in mammals that are expressed in nearly all cell downstream effectors of Rac is PAK (p21-activated types: the soluble and the membrane bound GCs (Lucas kinase). PAKs are a highly conserved family of serine/ et al., 2000). The soluble GCs are generally activated threonine protein kinases (Bokoch, 2003). Nearly all when nitric oxide (NO) binds to the attached prosthetic eukaryotes contain one or more PAK genes (Hofmann heme group. Seven membrane bound GCs (also named Cell 128, 341–355, January 26, 2007 ª2007 Elsevier Inc. 341 transmembrane or particulated GCs) have been identified not increase the cellular cGMP levels when their con- in the human genome (Lucas et al., 2000). GC-A and GC-B stitutively activated mutant forms (RhoA(G14V) and are natriuretic peptide receptors. GC-C can be activated Cdc42(G12V)) were transiently expressed in CHO-GC-E by bacterial heat-stable enterotoxins, guanylin, and uro- cells (Figure 1A). The lack of stimulation by RhoA and guanylin. The extracellular ligands for GC-D, GC-E, GC-F, Cdc42 was not due to poor expression of these proteins and GC-G are not known. The activity of transmembrane in CHO cells (Figure 1A, bottom western blots). Further- GCs can also be modulated by other receptor signals more, expression of RhoA(G14V) and Cdc42(G12V) in through intracellular signaling pathways. GC-E and GC-F, fibroblast cells led to the formation of actin stress fibers found in the retina, can be modulated by a group of reti- and filopodia, respectively (Figure S1B), indicating that nal-specific cellular proteins named GCAPs (guanylyl these expressed proteins were functional. cyclase activating proteins) in a calcium-dependent man- To examine whether Rac1 regulation of GC-E is exten- ner (Palczewski et al., 1994). Moreover, in genetic studies sive to other GCs, we tested the effect of Rac1 on the of olfaction in C. elegans, mutants defective in olfaction activity of GC-A, GC-D, GC-F, and the a1b1 soluble GC. sensory response have been obtained including daf-11 Rac1(G12V) was transiently expressed in cells stably and odr-1, two transmembrane GCs (Birnby et al., 2000; expressing GC-A, GC-D, GC-F, or soluble GC. As shown L’Etoile and Bargmann, 2000). Reintroduction of the intra- in Figure 1B, Rac1(G12V) increased the activity of GC-A, cellular domain of odr-1 rescued the defective phenotype GC-D, and GC-F. Rac1 had no effect on the activity of (L’Etoile and Bargmann, 2000). Transmembrane GCs soluble GC even though the cells expressing soluble GC function in regulating cell migration and actin cytoskeletal responded to the soluble GC activator sodium nitroprus- reorganization. The sea urchin sperm membrane bound side (Figure 1B). Furthermore, neither RhoA(G14V) nor GC is critical for sperm chemotaxis to the eggs (Bentley Cdc42(G12V) had any effect on GC-D activity (Figure 1B). et al., 1986). In addition, Dictyostelium mutants lacking These data indicate that Rac1 regulation of transmem- GCs are defective in cell chemotaxis (Bosgraaf et al., brane GCs is a general phenomenon. 2002). Furthermore, the Drosophila transmembrane GC To verify that membrane signaling receptors could use Gyc76C has been genetically demonstrated to be essen- this Rac-mediated pathway to modulate cellular cGMP tial for axonal guidance (Ayoob et al., 2004). levels, we tested PDGFR as a representative of the growth Although various types of membrane signaling recep- factor receptor tyrosine kinases. In serum-starved CHO- tors such as growth-factor receptor tyrosine kinases can GC-D cells, PDGF treatment led to higher cellular cGMP increase cellular cGMP levels (Coffey et al., 1988; Schev- levels (Figure 1C); this increase is comparable to that ing et al., 1985), the molecular mechanism by which these induced by Rac1(G12V) in CHO-GC-D cells (Figure 1B). receptors increase cGMP is not known. Here we have dis- Furthermore, expression of a dominant-negative Rac1 covered a new signaling pathway linking the small GTPase mutant (Rac1(T17N)) blocked PDGF-induced increase of Rac to the increase of cellular cGMP. Our data show that cellular cGMP in CHO-GC-D cells (Figure 1C and transmembrane GCs can be directly stimulated by intra- Figure S1C). In addition, cellular cGMP accumulation cellular PAK kinases. Previous studies have shown that was observed beginning at 1 min after PDGF treatment second messengers such as cGMP and Rho-family small (Figure 1D), similar to Rac activation by PDGF (Itoh GTPases such as Rac regulate cell migration and actin et al., 2002). Moreover, in Rac1-deficient mouse embry- cytoskeletal reorganization. However, the link between onic fibroblast (MEF) cells, PDGF-induced increase of cGMP and Rac has, until now, been missing. Our finding cellular cGMP was blocked (Figure 1E). Re-expression bridges these two important regulators of cellular physio- of Rac1 in these Rac1-deficient cells restored the cGMP logical functions. response (Figure 1E). These data demonstrate that Rac relays the PDGF signal to increase GC activity. RESULTS PAK Mediates Rac and PDGF Effect on Cellular Rac Increases Cellular cGMP Levels cGMP Levels In our