IQGAP1: A Regulator of Intracellular Spacetime Relativity Subramaniam Malarkannan,*,† Aradhana Awasthi,* Kamalakannan Rajasekaran,* Pawan Kumar,* Kristina M. Schuldt,* Allison Bartoszek,* Niranjan Manoharan,* Nicholas K. Goldner,* Colleen M. Umhoefer,* and Monica S. Thakar*,‡ Activating and inhibiting receptors of lymphocytes col- signaling molecules to achieve optimal functional outcomes. lect valuable information about their mikro`sko´smos. Cytoplasmic scaffolding such as IQGAP, Carma1, This information is essential to initiate or to turn off KSR1, Ste5, MP1 Paxillin, or PSD-95 can function as pro- complex signaling pathways. Irrespective of these advan- cessing centers of kinases and their substrates. IQGAP1 is ces, our knowledge on how these intracellular activation one of the most evolutionarily conserved (.90%) scaffolding cascades are coordinated in a spatiotemporal manner is proteins and is present in a variety of organisms. There are far from complete. Among multiple explanations, the three isoforms of IQGAPs (1, 2, and 3) that are described in scaffolding proteins have emerged as a critical piece of human and mouse. Among these, IQGAP1 is ubiquitously this evolutionary tangram. Among many, IQGAP1 is found (2), whereas the expressions of IQGAP2 (liver, platelets, one of the essential scaffolding proteins that coordinate kidney, stomach, prostate, thyroid, and salivary glands) (3–6) multiple signaling pathways. IQGAP1 possesses multi- and IQGAP3 (brain, lung, testis, small intestine, and colon) (3, 7) are restricted. Among lymphocytes, NK (8), B, and ple interaction motifs to achieve its scaffold- T cells (S. Malarkannan, unpublished observations) predom- ing functions. Using these domains, IQGAP1 has been inantly express IQGAP1. This review abridges the recent ex- shown to regulate a number of essential cellular events. citing information on IQGAP1 and its cellular functions (8). This includes polymerization, tubulin multime- rization, microtubule organizing center formation, cal- Protein structure and cellular partners of IQGAP1 scaffolding protein cium/ signaling, Pak/Raf/Mek1/2-mediated Erk1/2 activation, formation of maestrosome, E-cadhe- IQGAP1 is a 190-kDa protein, and its functional domains rin, and CD44-mediated signaling and glycogen syn- indicate that it is a critical regulator of development and thase kinase-3/adenomatous polyposis coli-mediated functions in multiple cell types. It was identified in 1994 as a widely expressed IQ domain-containing protein (9). This b-catenin activation. In this review, we summarize the 1657-aa long scaffolding protein has been described to asso- recent developments and exciting new findings of cellu- ciate with .50 different protein partners (10). Knockout lar functions of IQGAP1. The Journal of Immunology, mouse for IQGAP1 has been generated, where the null 2012, 188: 2057–2063. mutants bred at normal frequencies and no other major physiologic defects could be identified except a significant ncessant communications of immune cells with their onset of gastric later in their life (11). A possible environment govern their development, trafficking, rec- explanation for these observations could be the presence I ognition of target Ags/cells, phenotypic conversion from of IQGAP2 and IQGAP3 isoforms. Multiple cell surface effector to memory, or apoptotic death. Recent studies indicate receptors have been described to directly recruit IQGAP1 the bygone era of linear signaling pathways and a paradigm through their cytoplasmic tails, including . The shift to complex network circuitries. Integration of “spacetime E- and N-cadherins recruit IQGAP1 to maintain their trans relativity” is obligatory to execute intended biological func- interactions in the tight and adherens junctions (12). E- tions in lymphocytes. Multiple mechanisms have been de- cadherins are primarily expressed on epithelial cells but their scribed that facilitate the spatiotemporal coordination of sig- ability to interact with receptors such as KLRG1 (13–18) that naling events in lymphocytes (1). Among these, scaffolding has been shown to play a critical role in the proteins play an important role. Scaffolding proteins are de- of NK cells (19) further emphasize the potential roles of fined as proteins that can recruit, coordinate, and facilitate the IQGAP1 in the immune system. Recent studies show that E- spatiotemporal organization and the sequential activation of also interacts with integrin CD103 (20–22) that is

*Laboratory of Molecular Immunology and Immunotherapy, Blood Research Institute, Address correspondence and reprint requests to Dr. Subramaniam Malarkannan or Dr. Milwaukee, WI 53226; †Department of Medicine, Medical College of Wisconsin, Mil- Monica S. Thakar, Room 2015, Blood Research Institute, 8727 Watertown Plank Road, waukee, WI 53226; and ‡Department of Pediatrics, Medical College of Wisconsin, Milwaukee, WI 53226-2178 (S.M.) or Medical College of Wisconsin, MACC Fund Milwaukee, WI 53226 Research Center, Suite 3018, 8701 Watertown Plank Road, Milwaukee, WI 53226 (M.S.T.). E-mail addresses: [email protected] (S.M.) and [email protected] (M.S.T.) Received for publication December 1, 2011. Accepted for publication January 6, 2012. Abbreviations used in this article: CH, homology; GTPgS, guanosine 59-(3-O- This work was supported in part by National Institutes of Health Grant R01 A1064828 thio)triphosphate; Ras-GAP, Ras GTPase-activating protein. (to S.M.), the Hyundai Scholars Program (to M.S.T.), the Rebecca Jean Slye Endow- ment (to M.S.T.), the Midwest Athletes against Childhood Fund, and by the Ó Clinical and Translational Science Institute of Southeastern Wisconsin (National Insti- Copyright 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 tutes of Health Grant UL1RR031973) (to M.S.T.). www.jimmunol.org/cgi/doi/10.4049/jimmunol.1102439 2058 BRIEF REVIEWS: ROLE OF IQGAP1 AS A SIGNAL REGULATOR expressed in specific T cell subsets (20, 23, 24) and CD11c- high dendritic cells (25). These studies provide a functional framework of how E-cadherin on mucosal epithelia or thy- mus can influence the maturation and migration of CD103+ T cells and dendritic cells. Additional future works are re- quired to precisely determine the role of IQGAP1 in these cell–cell interactions. In a recent study, IQGAP1 has been shown to be part of a large cytoplasmic complex that con- tained phosphorylated NFAT1, long intergenic noncoding RNA, noncoding (RNA) repressor of NFAT (NRON), and three of the kinases that are responsible for NFAT1 phos- phorylation (26). This study further showed that the IQGAP1 preferentially interacted with phosphorylated NFAT1. Lack of IQGAP1 increased the dephosphorylation of NFAT1, its entry into the nucleus resulting in an augmented production of IL-2 or IFN-g from T cells after mitogenic activation (26). These results, while defining the potential scaffold functions of intergenic noncoding RNA, also emphasize the novel func- tions mediated by IQGAP1 in immune cells. IQGAP1 is composed of multiple protein recognition motifs (Fig. 1A). As the name denotes, a unique domain in IQGAP1 contains to the Ras GTPase-activating proteins (Ras-GAP). However, this domain lacks GAP activ- ity and thereby is unable to regulate Ras-GTP or Rap-GTP hydrolysis. The N-terminal calponin homology (CH) domain of IQGAP1 binds to actin (27, 28), whereas the IQ domain recruits calmodulin (29). The WW domain, with two highly conserved tryptophans, is an interaction module for proline- FIGURE 1. Protein structure and interacting partners of IQGAP1. (A) rich ligands (30) and binds to Erk1/2 (31). The functional role IQGAP1 is a 190-kDa protein that contains at least six distinct protein- of the coiled–coil domain is not known; however, it has a interacting domains. The calponin homology (CH) domain binds to poly- presumptive a-helical domain with significant sequence ho- merized F-actin. The function or the interacting partners of the coiled-coil (CC) domain has yet to be defined. The two highly conserved tryptophan- mology to myosins. containing (WW) domains recruit Erk1/2. Isoleucine/glutamine-containing The IQ domain is a tandem repeat of four IQ motifs (IQ) domain is a binding domain for multiple proteins including Rap1a, that mediates interactions with MEK1/2, myosin essential Rap1b, Mek1, Mek2, myosin ELC, Rafs, , and Ca2+-independent light chains (9), S100B (a Zn2+- and Ca2+-binding protein) interaction of calmodulin and its related proteins. Ras-GAP domain (GRD) (32), calmodulin (27, 29, 33), and calmodulin-related pro- interacts with small GTPases Cdc42 and Rac1. Ras-GAP C terminus domain (RGCT) interacts with the microtubule-binding protein Clip-170, b-catenin, teins (Fig. 1A) (34). Pathmanathan et al. (35) demonstrated B that the first IQ domain recruited myosin essential L chain E-cadherin, Clasp2, and adenomatous polyposis coli. ( ) The CH domain of IQGAP1 interacts with polymerized F-actin. Furthermore, IQGAP1 can Mlc1sa, whereas the first and the fourth interacted with my- delay the hydrolysis of Cdc42 and stabilize its interaction with WASp/Arp2/3 osin L chain, Mlc1p, from yeast. The first and second IQ complex. IQGAP1 also plays important roles in linking actin meshwork with domains were responsible for interacting with S100B. The plus-ends of microtubules through Clasp2. The role of IQGAP1 in the for- C-terminal end of IQGAP1 engages with Cdc42-GTP (29), mation and function of MTOC is not well understood. Both Clip-170 and Rac1-GTP (33), E-cadherin (12), b-catenin (36), and ade- APC may facilitate the organization and multimerization of g-tubulin mol- nomatous polyposis coli (37). IQGAP1 also has the ability to ecules that form the core of the centrosome and MTOC. Confocal images of NK cells are shown. Original magnification 3100. bind to Rap1 (38), B-Raf or C-Raf (39), Mek1/2 (40), and Erk1/2 (31). Ras-GAP domain (GRD) interacts with small GTPases such as Cdc42 (29, 33), Rac1 (33), and TC10 (41). critical cellular functions of IQGAP1, the immunological This GAP domain lacks the ability to hydrolyze the bound relevance of many of these protein interactions and the ef- GTP. Crystal structure of this region indicates that the fector regulations by IQGAP1 has yet to be defined. GRD domain of IQGAP family possesses a conserved thre- onine instead of the catalytic “arginine finger” described in IQGAP1 regulates actin/tubulin and microtubule functional Ras GAPs that is obligatory for GTP hydrolysis organizing center formation (42). The Ras-GAP C terminus domain interacts with the The actin cytoskeletal structure functions as a regulator of microtubule-binding protein Clip170 (43), b-catenin (44), E- signaling and cell integrity. IQGAP1 can directly interact with cadherin (12), adenomatous polyposis coli, and Clasp2 (Fig. F-actin and regulate the actin meshwork formation (6,9, 27, 1A) (37). Other proteins such as AKAP79 have been shown to 28, 43, 48–83). In particular, the N-terminal CH domain of regulate calcium flux via PKA by directly binding to a C- IQGAP1 directly interacts with the actin meshwork (Fig. 1B) terminal domain of IQGAP1 (45, 46). Thus, IQGAP1 can (37, 84). This CH motif of IQGAP1 and IQGAP2 are similar potentially regulate cell polarization, transcription, actin and to the F-actin–binding domains present in members of the microtubule function, MAPK cascade, and Ca2+/calmodulin spectrin, filamin, and fimbrin families. Detailed biochemical signaling (47, 48). Although these studies demonstrate the analyses have revealed that IQGAP1 using its CH domain The Journal of Immunology 2059 interacts with F-actin in a way that is critical to regulate the IQGAP1 plays a critical role in the regulation of tubulin polymerization of actin (28, 52). It has been demonstrated that multimerization and microtubule remodeling, its unique role the purified IQGAP1 can directly bind to F-actin and cross- on microtubule organizing center (MTOC) is not well un- link the actin filaments into irregular, interconnected bundles derstood. Evidence on the role of IQGAP1 on MTOC re- that exhibit gel-like properties (85). In addition, IQGAP1 can orientation came from studies by Watanabe et al. (37). Re- also interact with various proteins that are involved in cyto- cent studies from the Malarkannan laboratory demonstrate skeletal reorganization (43, 52, 69). This includes Cdc42 and that the size and shape of MTOC could be regulated by Rac1 (33, 49, 85), adenomatous polyposis coli (86), CLIP-170 IQGAP1 via the small GTPase, Rap1b (8). Rap1b has been (87), Clasp2 (88), and EB1 (89). Adenomatous polyposis coli shown to directly interact with IQGAP1 (38). Lack of Rap1b that has been well characterized to regulate the polarized cell did not affect the formation of the MTOC. However, the size migration can also directly interact with IQGAP1 (37). and the length of MTOCs were proportionately much larger Cdc42 and Rac1 belong to the Rho family of small guano- in NK cells that lacked Rap1b. Lack of Rap1b results in re- sine-3-phosphatases and play a significant role in regulating duced ERK1/2 phosphorylation primarily because of an im- the cellular cytoskeleton (43, 90). In recent years, the require- pairment in the sequential phosphorylation of B-Raf/C- ment of IQGAP1 in Cdc42 and Rac1-mediated actin poly- Raf→MEK1/2→ERK1/2 signaling pathway that requires merization has been well established. Indeed, the GRD domain the presence of the IQGAP1 scaffold (8). Other studies of IQGAP1 directly recruits small GTPases such as Cdc42 (29, by Kanwar et al. (92) demonstrate that the knockdown of 33), Rac1 (33), and TC10 (41). Importantly, Cdc42-GTP and IQGAP1 in the NK cell line YTS resulted in the inability of Rac1-GTP but not RhoA (6, 85) or Ras (33) have been shown MTOC to reorient, whereas the ability of YTS cells to form to interact with the GRD domains of IQGAP1 and IQGAP2. conjugates with target cells was preserved. Lack of IQGAP1 Thus, it appears that the activated Cdc42-GTP will function as did not grossly affect the development of T cells in the thy- a linker between the WASp/Arp2/3 complex and IQGAP1/ mus. However, in the absence of IQGAP1, T cells fail to APC/Clip170/Clasp2 complexes. The ability of the N- accumulate F-actin or polarize their MTOC toward anti– terminal region of IQGAP1 (1–216 aa) to directly interact CD3-coated beads (93). with F-actin (49) brings additional questions on the precise functional role played by IQGAP1. Is IQGAP1 critical for IQGAP1 provides the scaffold for the sequential phosphorylation of → → → forming the actin meshwork using polymerized actin filaments? Pak Raf Mek Erk1/2 The ability of IQGAP1 to cross-link actin was augmented Activation and phosphorylation of Erk1/2 constitute a key by guanosine 59-(3-O-thio)triphosphate (GTPgS).GST-Cdc42 signaling event in all lymphocyte subsets. Erk1/2 phosphor- but not by GDP.GST-Cdc42 (91). This augmentation oc- ylation regulates development and effector functions of T, B, curred by a preferential oligomerization of IQGAP1 by and NK cells (8, 94, 95). MAPKs, in particular Erk1/2, have GTPgS.GST-Cdc42. These findings reveal that the oligo- an important role in regulating the generation of IFN-g, GM- merization of IQGAP1 is a crucial step mediated by Cdc42 in CSF, MIP-1a, MIP-1b, and RANTES and cytotoxicity in NK regulating the cross-linking of filamentous b or g actin (49). cells (8, 96). MAPKs have also been shown to play a central role Other quantitative colocalization studies have shown that in the cytotoxic granule exocytosis from NK cells (97, 98). IQGAP1 also plays a significant role in the colocalization of Therefore, their phosphorylation and subsequent subcellular N-WASp in close proximity to Arp2/3 complex in lamelli- compartmentalization has to be tightly regulated to achieve podial structures (84). In addition, coimmunoprecipitation, intended outcomes. Multiple scaffolding proteins including pull-down and kinetic assays demonstrate that the C-terminal IQGAP1 (31, 99), KSR1 (100, 101), MP1 (102, 103), and half of IQGAP1 activated N-WASp by interacting with its b-arrestins (104–107) have been shown to regulate Erk1/2 BR-CRIB domain similar to that of Cdc42, while the N- activation in lymphocytes. Recent studies by Awasthi et al. terminal half of IQGAP1 antagonizes this activation by as- (8) demonstrate that IQGAP1 plays an important role in sociation with a C-terminal region of IQGAP1 through intra- regulating the Rap1b-GTP→Vav1→Cdc42→Pak→B-Raf/C- molecular interactions (84). Thus, a structural change in the Raf→Mek1/2→Erk1/2 signaling pathway in NK cells (Fig. 2). IQGAP1 protein can function as an autoregulatory switch Through its scaffolding function, IQGAP1 has the ability to that when turned “on” can activate N-WASp resulting in the recruit and sequentially regulate the activations of B-Raf (39), stimulation of actin assembly in an Arp2/3-dependent man- Mek1/2 (40), and Erk1/2 (31). Thus, IQGAP1 can play a sig- ner. The shape and morphology of dendritic arbors of neu- nificant role in the cytokine/chemokine transcriptions rons depend on the plus-end tracking protein Clip-170 and and other effector functions of lymphocytes. Each member of IQGAP1 (81). This study demonstrates that a direct inter- this sequential phosphorylation of protein kinase cascade, action of mTOR kinase with Clip-170 is required for the namely B-Raf, Mek1/2, and Erk1/2, have been shown to bind formation of the Clip-170/IQGAP1 complex. This complex directly to IQGAP1 in vitro and in intact cells (12). is capable of regulating the actin/tubulin in At the start of this sequence, receptor-mediated signaling primary hippocampal and cortical neurons. Thus, IQGAP1 activates Fyn, a Src family protein tyrosine kinase, to phos- can function as a focal point for feedback interactions between phorylate Vav-1 (108). Activated Vav-1 is one of the major the actin and microtubule cytoskeletal systems. One of the GEFs required for Cdc42 or Rac1 activation that results in first evidences that IQGAP1 could play a major role in the the conversion of GDP into GTP forms. The active Cdc42- reorganization of cytoskeleton came from Kaibuchi’s labora- GTP uses its second b-strand and a region of a peptide loop tory (43). This study showed that the activated Rac1/Cdc42, between the first a-helix and switch I region to bind the IQGAP1, and CLIP-170 form a tripartite complex. Although PBD46 motif of Pak with very high affinity (109). Studies these studies provide a mechanistic explanation of how have also indicated that Cdc42 exhibits differential binding 2060 BRIEF REVIEWS: ROLE OF IQGAP1 AS A SIGNAL REGULATOR

Erk2 is predominantly recruited and phosphorylated via this sequential activation (31).

IQGAP1 forms a master signalosome Mechanistic insights of how IQGAP1 functions as a scaf- folding protein during Erk1/2 activation or b-catenin–me- diated gene transcriptions are fundamental in understanding signaling processes in lymphocytes. More importantly, the transient spatiotemporal organization of the signaling events by IQGAP1 will provide novel insights and will help to de- velop additional cellular paradigms. Recently, Awasthi et al. (8) have demonstrated that IQGAP1 can form a unique sig- nalosome in the perinuclear region of NK cells to coordinate the phosphorylation of Erk1/2 (Fig. 3). A significant quantity of phospho-ERK1/2 was colocalized with IQGAP1 in these activated NK cells. This novel IQGAP1-mediated signalo- some enforced abundant but transient phosphorylation of Erk1/2 and thereby the effector functions of NK cells. In FIGURE 2. IQGAP1 scaffold regulates Erk1/2 phosphorylation. Sequential nonstimulated NK cells, phospho-Erk1/2 was not detectable, activation of Cdc42-Pak1-B/C-Raf-Mek1/2-Erk1/2 occurs on the IQGAP1 scaffold. The GRD domain, the four IQ repeats, and the WW domain of the and the IQGAP1 was distributed throughout the IQGAP1 are involved in the recruitment and phosphorylation of Cdc42, Rafs, with slight accumulation around the perinuclear region. After and Mek1/2 and Erk1/2, respectively. Membrane proximal signaling from 15 min of activation, Erk1/2 phosphorylation was evident in activation receptors activate the small GTPase Cdc42 that is recruited to the NK cells, and after 30 min of activation, the distribution GRD domain of IQGAP1. This in turn results in the recruitment and activation pattern of an IQGAP1 was drastically altered with a strong of Pak1. Although no direct binding of Pak1 to IQGAP1 has been described, it accumulation around the nucleus in NK cells. can bind to Rafs, and it is critical for the conformational change and activation of Both nonstimulated and NKG2D-activated NK cells con- B- or C- Rafs. Rafs are recruited to the IQ domains of the IQGAP1. Activation of Rafs results in the recruitment and phosphorylation of Mek1/2 to the IQ tained comparable levels of total ERK1/2 proteins (8). It is also domains. Activated Mek1/2 is essential for the recruitment and phosphorylation important to note that IQGAP1 can oligomerize to form of Erk1/2 that have the ability to bind to the WW domains of the IQGAP1. macromolecular structures (118). This study demonstrates that IQGAP1 exists as a combination of monomers, dimers, and patterns to Pak1, WASp, and IQGAP1 (110). Independently, it has been shown that the activated Cdc42-GTP used the “insert region” (111) and a part of the switch I domain (112) to interact with IQGAP1. Specifically, the switch I domain (aa 29–55) served as the binding site for Pak1, whereas the determinants outside this region (aa 84–120 and 157–191) were required for the bind- ing of IQGAP1 (and WASp). Synthetic peptide analogs from the PBD46 motif of Pak partly prevented the ability of Cdc42 binding to IQGAP1. This demonstrates IQGAP1, Pak, and WASp may form complexes that may interact with Cdc42 in a synchronized fashion (112). The next substrate in the se- quential activation on the IQGAP1 scaffold is Raf, which re- quires the direct binding of Pak1 for inducing conformational changes and phosphorylation of Ser338 (Raf-1) or Ser445 (B- Raf) (113, 114). Under resting conditions, both Raf-1 and B- FIGURE 3. Role of IQGAP1 in maestrosome formation. (A) IQGAP1 can Raf contain an N-terminal autoinhibitory domain that interacts compartmentalize and coordinate the signaling processes of multiple activa- with its catalytic domain (115, 116). However, upon activation, tion cascades that include the MAPK and b-catenin/TCF/LEF pathways. H-Ras (116) or another Ras family member, Rap1b (8), can Evidence indicates that during the activation of MAPK pathway, IQGAP1 directly interact with Rafs and relieve this autoinhibition. forms large macromolecular scaffolding structures. We name this IQGAP1- based master signalosome “maestrosome” to denote its macromolecular size. Although, these detailed studies explain the mechanism of (B) Activation of NK cells through NKG2D and the resulting Erk1/2 Raf activations, the temporal kinetics of how Cdc42/Pak1 phosphorylation provide proof-of-principle for the formation of a maestro- complex transfers Pak1 to B- or Raf-1 or the precise se- some around the nucleus. (C) Confocal images of NK cells 30 min post– quence in which small GTPases mitigate their autoinhibition NKG2D-mediated activation. These cells were stained for IQGAP1, phos- on the IQGAP1 scaffold have yet to be determined. B- or Raf-1 pho-Erk1/2, and nuclei (DAPI). Before activation, IQGAP1 was distributed phosphorylate Mek1/2, which has been shown to be recruited throughout the cytoplasm (Ref. 2; data not shown). However, after activation, the distribution pattern of an IQGAP1 was drastically altered with a strong to the IQGAP1 scaffold (40). IQGAP1-null cells are unable to accumulation around the nucleus. In addition, considerable quantities of augment the EGF-mediated Raf phosphorylation of Mek1/2 phospho-Erk1/2 colocalized around IQGAP1. These findings demonstrate confirming the critical role of this scaffold (39, 117). Al- the role of IQGAP1 in the formation of a maestrosome around the nucleus though both Mek1 and Mek2 equally bind to IQGAP1, only that regulates the MAPK pathway. The Journal of Immunology 2061 larger oligomers. The self- association region was mapped to 9. Weissbach, L., A. Bernards, and D. W. Herion. 1998. 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