Scaffold proteins dictate Rho GTPase-signaling specificity

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Scaffold proteins dictate Rho GTPase-signaling specificity Maria Julia Marinissen1 and J. Silvio Gutkind2 1 Instituto de Investigaciones Biomedicas A. Sols UAM-CSIC, Departamento de Bioquimica, Facultad de Medicina, Universidad Autonoma de Madrid, Arzobispo Morcillo 4, Madrid 28029, Spain 2 Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, 30 Convent Drive, Bethesda, MD 20892, USA

Given the numerous mechanisms that regulate the activity of Rho GTPases and the multiple effectors for Rho proteins, how is specificity achieved when transducing signals via Rho GTPase-regulated molecular networks? The finding that the scaffold protein hCNK1 links Rho guanine-nucleotide-exchange factors and Rho to JNK (c-Jun N-terminal kinase), while limiting stressfiber formation and serum-response-factor activation, suggests that scaffold proteins govern the selection of signal outputs, thus helping to solve the Rho GTPasesignaling puzzle. Rho-GTPase signaling networks The Rho family of small GTPases, including RhoA, Rac1 and Cdc42, control numerous cellular processes ranging from the regulation of the actin-based cytoskeleton, cell morphology and motility, to nuclear gene expression, and normal and aberrant cell growth. They function as molecular switches that are inactive when bound to GDP, often in association with GDP-dissociation inhibitors, and are active when GDP is exchanged for GTP upon interaction with their specific guanine-nucleotideexchange factors (GEFs) [1]. Signaling is terminated by the hydrolysis of GTP to GDP, a process accelerated by GTPase-activating proteins (GAPs). To date, O70 GEFs and O80 GAPs have been identified; their high number enables a wide variety of cell-surface receptors to control the activity of Rho proteins. In turn, Rho GTPases interact with a highly diverse set of target molecules, ranging from protein and lipid kinases to phospholipases, adaptor molecules and cell-surface receptors [1], thus acting as key nodes for signal integration and dissemination. Given the multiplicity of molecules that converge to regulate the activity of Rho GTPases and the diversity of their effectors, it is surprising that Rho proteins can exquisitely orchestrate the appropriate cellular response to each extracellular cue. In this regard, work by Buchsbaum et al. [2,3] and a recent study by Jaffe et al. [4] demonstrate that the interaction of GEFs with scaffold proteins can dictate the choice of downstream targets for Rho proteins. These findings provide new clues to how signal specificity and fidelity can be achieved when transducing signals via Rho GTPase-regulated molecular networks. Corresponding authors: Marinissen, M.J. ([email protected]), Gutkind, J.S. ([email protected]). Available online 5 July 2005 www.sciencedirect.com

Rho GTPases regulate the cytoskeleton and gene expression In the early 1990s, Rho GTPases emerged as master regulators of the spatio-temporal organization of intracellular pools of polymerized actin [1]. Surprisingly, two Rho family members, Rac and Cdc42, were later shown to regulate gene expression by activating c-Jun N-terminal kinase (JNK), which is a member of the mitogen-activated protein kinase (MAPK) group of proline-targeted serine/ threonine protein kinases [5]. These two events seemed to be highly inter-related, as constitutively activate mutants of Rac can induce both actin reorganization and JNK activity [6]. However, when endogenous Rac was stimulated by its upstream activators this was not necessary the case, as, for example, phosphoinositide-3 kinase (PI3K)-activated Rac induces actin polymerization but not JNK activation [7]. Furthermore, certain GEFs for Rac and Cdc42 stimulate Pak-1, an effector for both GTPases that promotes the localized polymerization of actin, whereas other GEFs favor JNK activation [8]. This raised the possibility that mechanisms might exist that ensure signaling specificity by selecting the appropriate targets for Rho GTPases in each distinct biological context. In this regard, evidence has emerged that intracellular pathways are often organized into functional signaling modules by scaffold proteins that interact with, and tether, key components of each signaling route. This was first documented in a study of the mating response to pheromones in yeast, in which a scaffold protein Ste5p, was found to be crucial for ensuring the timely and localized activation of a MAPK module by interacting with Ste11p (a MAPK kinase kinase), Ste7p (a MAPK kinase) and Fus3p (a MAPK) [9]. Scaffold proteins organizing MAPK-signaling modules, including extracellular-signaling regulated kinases (ERKs), p38s and JNKs, have also been described in mammals and other multicellular organisms [10]. Among them, the most extensively investigated are the members of the JNK-interacting proteins (JIP) family, JIP1–JIP4, that participate in the activation of JNK and p38 MAPK modules by virtue of their ability to assemble molecular complexes with their regulating molecules and upstream kinases [10]. The physical interaction with scaffold proteins might represent a general mechanism by which GEFs can govern the selection of signal outputs upon Rho GTPase activation. Indeed, the RacGEFs Tiam1 and Ras-GRF1 can bind to the scaffold protein JIP2, leading to the selective stimulation

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Figure 1. Scaffold proteins govern the selection of signal output upon Rac activation. PIX, a Rac-GEF, forms a molecular complex with the serine/threonine protein kinase Pak1. This facilitates the stimulation of Pak1 upon Rac activation, thereby, promoting the polymerization of actin, which results in rapid changes in the actin-based cytoskeleton and the formation of membrane ruffles known as lamellipodia. Two other guanine-nucleotide exchange factors for Rac (RacGEFs), Tiam1 and Ras-GRF1 (not shown), bind to the scaffold proteins JIP2 and JIP1, leading to the preferential activation of p38 and probably JNK by Rac, respectively, and the consequent phosphorylation of nuclear transcription factors that regulate gene expression. Arrows represent activation events either by direct binding or by phosphorylation as in the case of the kinases (orange) and transcription factors (brown).

of p38 upon Rac activation [2] (Figure 1). These GEFs also bind to JIP1 [2], which in turn assembles the JNK pathway [10], probably resulting in enhanced JNK activity in different cellular contexts. Tiam1 can also affect Rac-signaling specificity by interacting with spinophilin, a scaffold protein that binds to the p70 S6 kinase, thus facilitating the activation of this kinase [3]. Alternatively, Tiam1 can affect Rac-signaling specificity by binding to IRSp53 (insulin-receptor substrate 53), an adaptor protein that links Rac to WAVE2 (WASP verprolin-homologous 2), which is a scaffold protein that promotes the localized polymerization of actin in lamellipodia [11]. What dictates the selection of the protein scaffold to which Tiam1 binds in response to extracellular stimuli is still unknown. GEFs can also bind to kinases directly, as shown for PIX, a RacGEF that forms a complex with Pak1, thus favoring the stimulation of this kinase by bringing it into close proximity to GTP-bound Rac [12]. Therefore, GEFs can form molecular complexes with scaffold proteins and kinases to direct the preferential activation of selective signaling pathways upon Rac nucleotide exchange. hCNK1 as a scaffold for Rho-initiated pathways Whereas Rac and Cdc42 regulate nuclear events via JNK and p38, RhoA can stimulate expression from the c-fos serum-response element (SRE) by a MAPK-independent biochemical route [13,14]. This pathway involves the Rho-induced polymerization of actin into stress fibers, which ultimately results in the enhanced transcriptional activity of the serum-response factor (SRF) bound to the SRE [13,14] (Figure 2). However, recent evidence indicates that Rho can also control the activity of MAPK cascades, such as JNKs and p38s, which phosphorylate www.sciencedirect.com

the transcription factors c-Jun, ATF2 and MEF2 to regulate c-jun expression [15,16]. Unlike the examples described here for Rac, what influences the choice of signal output upon Rho activation is still not clearly understood. A recent study by Jaffe and colleagues show that the scaffold protein hCNK1 (human connector enhancer of ksr) links Rho and RhoGEFs to JNK activation and c-Jun phosphorylation, while limiting stress-fiber formation and SRF activation. These findings suggest a crucial role for hCNK1 in the selection of the available Rho-initiated pathways [4]. Connector enhancer of ksr (CNK1) was first isolated in a genetic screen in Drosophila to identify cofactors for kinase suppressor of ras (ksr), a positive regulator of Ras signaling [17]. Its human homolog, hCNK1, encodes a 720-amino-acid protein that contains a sterile a motif (SAM) domain, a PSD-95/DLG-1/ZO-1 (PDZ) domain, a pleckstrin homology (PH) domain and a region conserved in CNKs from multiple species (CRIC) [18]. Based on their prior observations that hCNK1 interacts with Rho and the Rho-target rhophilin [18], the authors investigated whether hCNK1 could associate with additional Rho-regulating molecules. In fact, they found that hCNK1 binds to the DH–PH (Dbl-homology–pleckstrin-homology) domain of two RhoGEFs, Net1 and p115-RhoGEF. Surprisingly, they observed that in HeLa cells hCNK1 overexpression diminishes stress-fiber formation and SRF activation while promoting c-Jun phosphorylation. As this process required the activation of Rho by p115-RhoGEF or Net1 but not the subsequent binding of hCNK1 to Rho, Jaffe et al. [4] hypothesized that hCNK1 might form a molecular complex linking RhoGEFs to components of the JNK pathway. They found that hCNK1 binds to two JNK kinase kinases, mixed-lineage kinase (MLK)-2 and MLK3,

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Figure 2. Scaffold proteins determine the signaling specificity of Rho. Cell-surface receptors coupled to the heterotrimeric G proteins Ga12 and Ga13 activate the small GTPase Rho via RhoGEFs, including p115-RhoGEF, LARG and PDZ-RhoGEF. In turn, Rho stimulates nuclear events and actin polymerization, often in a coordinated manner. The ability of the scaffold protein hCNK1 to form a molecular bridge between p115-RhoGEF, Rho, and the protein kinases MLK3 and MKK7, leads to the selective activation of JNK by Rho via this MAPK cascade. When activated by p115-RhoGEF in the absence of hCNK1, and by RhoGEFs that do not bind to hCNK1, Rho can instead stimulate JNK and certain p38 isoforms via distinct signaling modules, which include the ROCK/MKK4/JNK signaling axis and the MLTK/MKK6/p38 pathway, which phosphorylate nuclear transcription factors such as c-Jun, SRF and MEF2, thereby, regulating gene expression. In parallel, Rho promotes the remodeling of the cytoskeleton via ROCK and LimK, which inactivate an actin-severing protein, cofilin, thereby, enhancing actin polymerization and the consequent activation of the SRF independently of hCNK1. Arrows represent activation events either by direct binding or by phosphorylation as in the case of the kinases (red) and transcription factors (brown). Broken arrow represents putative interaction or activation. Question marks denote unknown molecules.

and the JNK kinase MKK7 but not MKK4. The formation of this multimeric protein complex might result in the stimulation of these MLKs, which are best known for being direct targets for Rac [19]. Also, in the presence of hCNK1, p115-RhoGEF co-immunoprecipitates with MKK7, which confirms that hCNK1 can function as a molecular bridge connecting RhoGEFs to the JNK pathway (Figure 2). Interestingly, the formation of a complex between p115-RhoGEF, hCNK1 and MLKs is required to induce c-Jun phosphorylation by sphingosine-1-phosphate (S1P), which activates p115-RhoGEF via Ga12/13. In fact, c-Jun phosphorylation, in response to S1P, can be blocked by hCNK1 knock-down without affecting stress-fiber formation. Notably, hCNK1-mediated JNK activation by Rho seems to be cell-type specific. For example, hCNK1 is undetectable in NIH 3T3 cells, but S1P can still activate JNK in these cells albeit to a limited extent [4]. In cells that do not express hCNK1, agonists that stimulate Rho, such as S1P and lysophosphatidic acid (LPA), might www.sciencedirect.com

instead activate JNK and c-Jun phosphorylation via an alternative pathway that involves the Rho-target ROCK (Rho-activated kinase), which stimulates MKK4 (but not MKK7) [16]. These findings provide excellent examples of how the repertoire of molecules available in each cell type can influence the selection of downstream targets for Rho GTPases to achieve a particular response. The role of hCNK1 as a protein scaffold that favors JNK activation might not be restricted to its effects on Rho and RhoGEFs. In fact, Jaffe and colleagues found that hCNK1 can also cooperate with Rac to activate JNK, suggesting that hCNK1 might interact with, as yet, unidentified RacGEFs. Furthermore, although Drosophila CNK was first described as a scaffold for the Ras/ERK pathway because it interacts with Drosophila Raf, an ERK kinase kinase, only one of its human homologs, hCNK2, can bind mammalian Raf [17,18,20]. Instead, both hCNK1 and hCNK2 interact with RalGDS and Rlf, respectively, which are GEFs for Ral, a Ras-related GTPase [18,20].

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This suggests that CNKs could have a more general role than expected because they might participate in the activation of JNK not only by Rho, but also by Ral [21], and even by Ras, which binds RalGDS [22]. Furthermore, because CNKs exhibit several structural motifs that are often involved in regulatory functions, these molecules and their functionally related scaffolding proteins might also represent potential nodes for signal integration. Concluding remarks The emerging notion from the studies highlighted here is that the activation of Rho GTPases can lead to distinct biochemical and biological responses depending on the nature of the intervening GEFs and the ability of these GEFs to interact with different scaffold proteins. Thus, scaffold proteins might function as key signal organizers that determine the specificity of target selection by Rho GTPases in a spatial-temporal manner to favor the activation of a distinct subset of downstream pathways. Highthroughput analysis of protein–protein interactions and the now widespread use of protein knock-down strategies, combined with cell-type-specific protein-expression studies and newly established real-time-imaging techniques might help us to understand what determines the choice of intervening GEFs and protein scaffolds in response to each extracellular cue, thus, helping unravel the complexity of the Rho GTPase-activated-signaling puzzle. References 1 Etienne-Manneville, S. and Hall, A. (2002) Rho GTPases in cell biology. Nature 420, 629–635 2 Buchsbaum, R.J. et al. (2002) Interaction of Rac exchange factors Tiam1 and Ras-GRF1 with a scaffold for the p38 mitogen-activated protein kinase cascade. Mol. Cell. Biol. 22, 4073–4085 3 Buchsbaum, R.J. et al. (2003) Regulation of p70 S6 kinase by complex formation between the Rac guanine nucleotide exchange factor (Rac-GEF) Tiam1 and the scaffold spinophilin. J. Biol. Chem. 278, 18833–18841 4 Jaffe, A.B. et al. (2005) Association of CNK1 with Rho guanine nucleotide exchange factors controls signaling specificity downstream of Rho. Curr. Biol. 15, 405–412 5 Coso, O.A. et al. (1995) The small GTP-binding proteins Rac1 and Cdc42 regulate the activity of the JNK/SAPK signaling pathway. Cell 81, 1137–1146

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6 Lamarche, N. et al. (1996) Rac and Cdc42 induce actin polymerization and G1 cell cycle progression independently of p65PAK and the JNK/SAPK MAP kinase cascade. Cell 87, 519–529 7 Reif, K. et al. (1996) Phosphatidylinositol 3-kinase signals activate a selective subset of Rac/Rho-dependent effector pathways. Curr. Biol. 6, 1445–1455 8 Zhou, K. et al. (1998) Guanine nucleotide exchange factors regulate specificity of downstream signaling from Rac and Cdc42. J. Biol. Chem. 273, 16782–16786 9 Elion, E.A. (2001) The Ste5p scaffold. J. Cell Sci. 114, 3967–3978 10 Morrison, D.K. and Davis, R.J. (2003) Regulation of MAP kinase signaling modules by scaffold proteins in mammals. Annu. Rev. Cell Dev. Biol. 19, 91–118 11 Connolly, B.A. et al. (2005) Tiam1–IRSp53 complex formation directs specificity of Rac-mediated actin cytoskeleton regulation. Mol. Cell. Biol. 25, 4602–4614 12 Daniels, R.H. et al. (1999) aPix stimulates p21-activated kinase activity through exchange factor-dependent and -independent mechanisms. J. Biol. Chem. 274, 6047–6050 13 Hill, C.S. et al. (1995) The Rho family GTPases RhoA, Rac1, and CDC42Hs regulate transcriptional activation by SRF. Cell 81, 1159–1170 14 Sotiropoulos, A. et al. (1999) Signal-regulated activation of serum response factor is mediated by changes in actin dynamics. Cell 98, 159–169 15 Marinissen, M.J. et al. (2001) Regulation of gene expression by the small GTPase Rho through the ERK6 (p38g) MAP kinase pathway. Genes Dev. 15, 535–553 16 Marinissen, M.J. et al. (2004) The small GTP-binding protein RhoA regulates c-jun by a ROCK-JNK signaling axis. Mol. Cell 14, 29–41 17 Therrien, M. et al. (1998) CNK, a RAF-binding multidomain protein required for RAS signaling. Cell 95, 343–353 18 Jaffe, A.B. et al. (2004) Human CNK1 acts as a scaffold protein, linking Rho and Ras signal transduction pathways. Mol. Cell. Biol. 24, 1736–1746 19 Burbelo, P.D. et al. (1995) A conserved binding motif defines numerous candidate target proteins for both Cdc42 and Rac GTPases. J. Biol. Chem. 270, 29071–29074 20 Lanigan, T.M. et al. (2003) Human homologue of Drosophila CNK interacts with Ras effector proteins Raf and Rlf. FASEB J. 17, 2048–2060 21 de Ruiter, N.D. et al. (2000) Ras-dependent regulation of c-Jun phosphorylation is mediated by the Ral guanine nucleotide exchange factor-Ral pathway. Mol. Cell. Biol. 20, 8480–8488 22 Hofer, F. et al. (1994) Activated Ras interacts with the Ral guanine nucleotide dissociation stimulator. Proc. Natl. Acad. Sci. U. S. A. 91, 11089–11093

0968-0004/$ - see front matter Q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibs.2005.06.006

HIF-1a and p53: the ODD couple? Diane R. Fels1 and Constantinos Koumenis1,2,3 1

Department of Cancer Biology, Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA Department of Radiation Oncology, Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA 3 Department of Neurosurgery, Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA 2

Tumor hypoxia activates hypoxia-inducible factor-1 (HIF-1) and induces the accumulation of the tumor Corresponding author: Koumenis, C. ([email protected]). Available online 5 July 2005 www.sciencedirect.com

suppressor p53. HIF-1 signaling stimulates angiogenesis and mediates cellular adaptation to hypoxia, whereas p53 promotes hypoxia-induced apoptosis. A recent article provides in vitro biophysical evidence supporting a direct interaction between p53 and the oxygen-dependent