E-Letters

Posted E-Letters:

• Correction to Sequence Range
  Science Signaling Editors (24 February 2012)
  sciencesignalingeditors@aaas.org
• Ras and Rap GAP Function and GTPase Sequestration in Plexin-Mediated Cell Signaling Mechanisms
  Matthias Buck, Manabu Negishi (8 March 2012)
  matthias.buck@case.edu
• Response to Negishi and Buck
  Xuewu Zhang, Yuxiao Wang, Huawei He, Nishi Srivastava, Sheikh Vikarunnessa, Yong-bin Chen, Jin Jiang, and Christopher W. Cowan (8 March 2012)
  xuewu.zhang@utsouthwestern.edu

Correction to Sequence Range

The authors have alerted the Science Signaling Editors that the sequence range of human PlexinB1 that was amplified by polymerase chain reaction was incorrectly stated in the Materials and Methods section. The correct sequence range is 1515-2135.

Ras and Rap GAP Function and GTPase Sequestration in Plexin-Mediated Cell Signaling Mechanisms

In a recent Science Signaling paper, Zhang and colleagues report the discovery of a Rap1 GAP function for plexins. This is an important paper. However, two aspects of the report require further study.

Ras GAP and Rap GAP function: Plexin transmembrane receptors function in diverse developmental and disease processes, including axon guidance, angiogenesis, and metastatic cell migration. The receptors are remarkable in that they interact with small GTPases at three levels: as effectors, binding to Rho GTPases; as regulators of Rho GTPase GAPs and GEFs; and as GAPs themselves. A GAP function towards Ras proteins was initially inferred from the homology of plexin intracellular regions to the Ras GAP family of regulatory proteins. GAP activity has been well established for members of all four human plexin families against R-Ras and M-Ras in cellular assays by five laboratories (1-3, for example), including by a biochemical assay using cell lysates (4). In these experiments, the Rho-family GTPase Rnd1 and clustering of plexin are required to activate plexin Ras GAP activity. Although R-Ras binding to the plexin-B1 intracellular region has been reported in vitro (5), it has been difficult to reconstitute the GAP activity for M- or R-Ras with purified protein components in solution (5, 6). Further work is clearly needed to successfully reconstitute the system.

Zhang and colleagues report a GAP function of plexin against Rap GTPases (6). In contrast to the situation with Ras, this Rap GAP activity is found in aqueous solution at high GTPase concentration. In particular, the intracellular domains of plexin-C1 and -D1 are functional, whereas plexin-B1 shows more modest activity. Activity could be stimulated in A-family plexins, and was increased in other plexins by induced N-terminal dimerization. Using constitutively active Rap1 protein, cell collapse was abolished in Sema3A-stimulated neurons, suggesting that the Rap GAP activity extends to cellular settings (6), but the signaling networks downstream of Rap in the case of plexins are not yet known [(7), for example]. Intriguingly, in vitro GAP activity seems to correlate inversely with the dimerization propensity of plexin family members themselves. For instance, plexin-A members can dimerize through their extracellular (8) as well as their intracellular domains (9, 10), whereas the Rho GTPase binding domains of plexin-C1 and -D1 are monomeric (10), suggesting that A- and B-family members can also engage in a dimer formation that inhibits Rap GAP activity. Overall the results suggest that plexins are multifunctional GAP proteins, similar to CAPRI, which is a dual specificity Ras/RAP enzyme (11). Similarly, we expect that different plexin GAP activities would also be controlled by different mechanisms and environments.

GTPase sequestration: The role and regulation of GTPase-plexin interactions also presents an ongoing area of intensive research with controversial findings and interpretations. Rho GTPases, especially Rnd1 and Rac1, can play an active role in receptor activation (1-4), although the structural mechanism is not yet clear, with experimental evidence supporting several, not necessarily exclusive models (3, 5, 6, 9). Structural perturbations in plexin crystallographic snap-shots that accompany GTPase binding may appear to be slight, but thermodynamic and mutagenesis studies suggest several binding modes and greater functional specificity for Rho GTPases than originally anticipated (12, 13, 14, 15). Intriguingly, Rap and Ras as well as several of the Rho GTPases also antagonize each other's function. Other modes of plexin-GTPase associations (referred to as sequestration) could also play a role. For example, as pointed out by Zhang and colleagues (6), there are specific cellular settings in which plexin binds R-Ras but, in absence of Rnd1, does not stimulate R-Ras GTP hydrolysis (16). This suggests a Ras sequestration model in which plexin may serve to withdraw the GTPase from the integrin system, thus diminishing cell adhesion in cell collapse. However, it is not clear whether sequestration provides an adequate control of Ras or Rho activity; in other systems compartmentalized or scaffold-bound proteins may still be active. Also, the binding affinity would need to be substantial and plexin concentration should be higher than that of the GTPases (12); neither of these requirements may be fulfilled in cells. Although sequestration is thought to have a larger impact on regulatory proteins or enzymes, recent studies also suggests that GTPase cycles are impacted when the GTPases or GTPase effectors are sequestered [for example, (17-20)]. The mechanisms are undoubtedly complex, but multilevel participation of plexins and GTPases is likely required in a temporally and spatially organized manner in order to create the waves of polymerization and depolymerization of the actin cytoskeleton, as well as integrin-mediated adhesion and de-adhesion, that are integral for cell migration. A "one fits-all" model, such as a general GTPase sequestration mechanism, is unlikely to work and much remains to be learned about GTPase-plexin interactions.

References

1. I. Oinuma, Y. Ishikawa, H. Katoh, M. Negishi, The semaphorin 4D receptor Plexin-B1 is a GTPase activating protein for R-Ras. Science 305, 862–865 (2004). [Abstract] [Full Text]


2. J. M. Swiercz, T. Worzfeld T, S. Offermanns, Semaphorin 4D signaling requires the recruitment of phospholipase C gamma into the plexin-B1 receptor complex. Mol. Cell Biol. 29, 6321-6334 (2009). [Abstract] [Full Text]


3. C. H. Bell, A. R. Aricescu, E. Y. Jones, C. Siebold, A dual binding mode for RhoGTPases in plexin signalling. PLoS Biol. 9, e1001134 (2011). [PubMed]


4. I. Oinuma, H. Katoh, M. Negishi, Molecular dissection of the semaphorin 4D receptor plexin-B1-stimulated R-Ras GTPase-activating protein activity and neurite remodeling in hippocampal neurons. J. Neurosci. 24, 11473-11480 (2004). [PubMed]


5. Y. Tong, P. K. Hota, J. Y. Penachioni , M. B.Hamaneh, S. Kim, R. S. Alviani, L. Shen, H. He, W. Tempel, L. Tamagnone, H.W. Park, M. Buck, Structure and function of the intracellular region of the plexin-b1 transmembrane receptor. J. Biol. Chem. 284, 35962-35972 (2009). [PubMed]


6. Y. Wang, H. He, N. Srivastava, S. Vikarunnessa, Y.B. Chen, J. Jiang, C.W. Cowan, X. Zhang, Plexins are GTPase-activating proteins for Rap and are activated by induced dimerization. Sci. Signal. 5, ra6 (2012). [Abstract] [Full Text]


7. A. Catalano, Caprari, P. Rodilossi, S. Betta P, M. Castellucci, A. Casazza, L. Tamagnone, A. Procopio, Cross-talk between vascular endothelial growth factor and semaphorin-3A pathway in the regulation of normal and malignant mesothelial cell proliferation. FASEB J. 18, 358-360 (2004). [PubMed]


8. T. Nogi, N. Yasui, E. Mihara, Y. Matsunaga, M. Noda, N. Yamashita, T. Toyofuku, S. Uchiyama, Y. Goshima, A. Kumanogoh, J. Takagi, Structural basis for semaphorin signalling through the plexin receptor. Nature 467, 1123-1127 (2010). [PubMed]


9. H. He, T. Yang, J.R. Terman, X. Zhang, Crystal structure of the plexin A3 intracellular region reveals an autoinhibited conformation through active site sequestration. Proc. Natl. Acad. Sci. U. S. A. 106, 15610-15615 (2009). [Abstract] [Full Text]


10. H. Wang, P.K. Hota, Y. Tong, B. Li, L. Shen, L. Nedyalkova, S. Borthakur, S. Kim, W. Tempel, M. Buck, H.W. Park, Structural basis of Rnd1 binding to plexin Rho GTPase binding domains (RBDs). J. Biol. Chem. 286, 26093-26106 (2011). [Abstract] [Full Text]


11. Y. Dai, S. A. Walker, E. de Vet, S. Cook, H. C. Welch, P. J. Lockyer, Ca²⁺-dependent monomer and dimer formation switches CAPRI Protein between Ras GTPase-activating protein (GAP) and RapGAP activities. J. Biol. Chem. 286, 19905-19916 (2011). [PubMed]


12. Y. Tong, P. Chugha, P. K. Hota, R. S. Alviani, M. Li, W. Tempel, L. Shen, H. W. Park, M. Buck, Binding of Rac1, Rnd1, and RhoD to a novel Rho GTPase interaction motif destabilizes dimerization of the plexin-B1 effector domain. J. Biol. Chem. 282, 37215-37224 (2007). [PubMed]


13. K. Uesugi, I. Oinuma, H. Katoh, M. Negishi, Different requirement for Rnd GTPases of R-Ras GAP activity of Plexin-C1 and Plexin-D1. J. Biol. Chem. 284, 6743-6751 (2009). [PubMed]


14. O. G-W. Wong, T. Nitkunan, I. Oinuma, C. Zhou, V. Blanc, R. S. D. Brown, Simon R. J. Bott, J. Nariculam, G. Box, P. Munson, J. Constantinou, M. R. Feneley, H. Klocker, S. A. Eccles, M. Negishi, A. Freeman, J. R. Masters, M. Williamson, Plexin-B1 mutations in prostate cancer. Proc. Natl. Acad. Sci. U. S. A., 104, 109040-10945 (2007). [PubMed]


15. C. Zhou, O. G.-W. Wong, J. R. Masters, M. Williamson, Effect of cancer-associated mutations in the PlexinB1 gene. Mol. Cancer 11, DOI: 10.1186/1476-4598-11-11 (2012).


16. A. Sakurai, J. Gavard, Y. Annas-Linhares, J. R. Basile, P. Amornphimoltham, T. R. Palmby, H. Yagi, F. Zhang, P.A. Randazzo, X. Li, R. Weigert, J. S. Gutkind , Semaphorin 3E initiates antiangiogenic signaling through plexin D1 by regulating Arf6 and R-Ras. Mol. Cell. Biol. 30, 3086-98 (2010). [PubMed]


17. N. Blüthgen, Sequestration shapes the response of signal transduction cascades. IUBMB Life 58, 659-663 (2006). [PubMed]


18. A. B. Goryachev, A. V. Pokhilko, Computational model explains high activity and rapid cycling of Rho GTPases within protein complexes. PLoS Comput. Biol. 2, e172 (2006). [PubMed]


19. A. Lipshtat, G. Jayaraman, J.C. He, R. Iyengar, Design of versatile biochemical switches that respond to amplitude, duration, and spatial cues. Proc. Natl. Acad. Sci. U. S. A. 107, 1247-1252 (2010). [PubMed]


20. M. A. Tsyganov, W. Kolch, B. N. Kholodenko, The topology design principles that determine the spatiotemporal dynamics of G-protein cascades. Mol. Biosyst. 8, 730-743 (2012). [PubMed]

Contributed by

Manabu Negishi
Kyoto University
E-mail: mnegishi@pharm.kyoto-u.ac.jp

Matthias Buck
Case Western Reserve University
E-mail: matthias.buck@case.edu

Response to Negishi and Buck

We are pleased that Drs. Manabu Negishi and Matthias Buck considered our work (1) important, and did not raise any specific criticisms of our data or interpretation.

As pointed out by Drs. Negishi and Buck, many small GTPases participate in plexin signaling. Some of them do so by direct interaction, others use indirect mechanisms. We agree that additional studies are required to reconcile some of the perplexing issues and elucidate these complicated signaling pathways, which should motivate future studies of people in the field, including ourselves.

Reference

1. Y. Wang, H. He, N. Srivastava, S. Vikarunnessa, Y.B. Chen, J. Jiang, C.W. Cowan, X. Zhang, Plexins are GTPase-activating proteins for Rap and are activated by induced dimerization. Sci. Signal. 5, ra6 (2012). [Abstract] [Full Text]

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