Drosophila Rho-associated kinase (Drok), an effector of RhoA, is essential for transducing signals to the actin cytoskeleton in wing cells (Winter, 2001). Since the effector domain mutant analysis of RhoA suggests that a cytoskeletal pathway is important for axon retraction, tests were performed to see if the Drok pathway is involved. Carboxy-terminal truncation of mammalian Rho-kinase/ROCK results in its constitutive activation. Expression of an analogous activated form (Drok-CAT; Winter, 2001) in MB neurons led to truncated dorsal lobes similar to the phenotypes of p190 RNAi and weak RhoA activation. A presumptive kinase-dead point mutation (Drok-CAT.KG; Winter, 2001) has no effect, indicating that Drok signaling is dependent on its kinase activity. Developmental studies indicate that the Drok-CAT phenotypes also results from axon retraction, as does the p190 RNAi phenotype (Billuart, 2001).
Neuroblast clones homozygous for Drok2 (Winter, 2001) do not show apparent defects in cell proliferation, because the adult clones contain dorsal axon lobes contributed by later born neurons. Close examination of Drok2 neuroblast clones reveals that 10 of 17 contain at least one axon that extends significantly further than the heterozygous neurons within the same MB. Although this phenotype is subtle, it is not seen in 19 control clones, the parental chromosome for the Drok2 mutant, nor in many other genotypes studied. Thus, it is concluded that Drok is required to limit dorsal axon extension (Billuart, 2001).
Biochemical and genetic evidence indicates that a key output for Drok signaling in vivo is the regulation of phosphorylation of myosin regulatory light chain (MRLC) encoded by spaghetti squash (sqh) (Winter, 2001). To test if endogenous MRLC is part of the axon retraction pathway regulated by p190, genetic interaction experiments were performed by reducing the dose of endogenous sqh in the context of the p190 dsRNA expression. Marked suppression of the phenotype was observed in flies heterozygous for a null mutation of sqh (sqhAX3). In contrast, expression of a phosphomimetic mutant, Sqh-E20E21, markedly enhanced the p190 phenotype, whereas analogous expression of a nonphosphorylable form (Sqh-A21) had no effect. Further, truncation of the medial lobe was frequently observed when Sqh-E20E21 was expressed with the intermediate p190 RNAi line. This is evident from the FasII staining, showing that the medial ß axons (strongly FasII positive) only extend a fraction of the length of the medial lobe. This phenotype was only observed in the strongest p190 RNAi lines, never in the intermediate line alone. Taken together, these results strongly suggest that Drok and phosphorylation of Drosophila MRLC participate in mediating axon retraction as a result of p190 inactivation (Billuart, 2001).
Since rat p190 can rescue the Drosophila RhoGAP (p190) loss-of-function phenotypes, tests were performed to see if upstream regulators of mammalian p190 could interact with Drosophila p190 to regulate MB axon morphogenesis. The Src family of tyrosine kinases phosphorylate mammalian p190. Tests were performed to see if the Drosophila Src homolog, Src64, regulates p190 activity. Heterozygosity for two Src64 alleles significantly suppresses the p190 RNAi phenotype, with the strength of suppression correlating with the strength of the alleles used. This result is consistent with the notion that Src64 negatively regulates p190 (Billuart, 2001).
Tests were performed to see if integrin could regulate p190 activity in MB neurons, since mammalian studies suggest potential links between integrin and p190, as well as integrin and Src. Integrins function as heterodimers of one alpha and one ß subunit. Drosophila has five genes for integrin alpha subunits, including one, volado (vol), also called scab, which is preferentially expressed in MB neurons and, when mutated, results in a short-term memory defect. No significant modification of the p190 RNAi phenotype was observed in flies heterozygous for a null mutation of vol (vol4). This may be because vol is not dosage sensitive or because it functions redundantly with other integrin alpha subunits in regulating p190 activity. Only two genes encode integrin ß subunits in the fly, ßPS and ßnu; ßnu may not associate with an alpha subunit. The myospheroid (mys) gene, encoding ßPS, shows robust genetic interaction with p190. In flies with one wild-type copy of mys, the p190 RNAi phenotype is markedly suppressed for each of the three mys alleles tested, suggesting that p190 is negatively regulated by integrin (Billuart, 2001).
Finally, MB neuroblast clones homozygous for mys1 were examined using the MARCM system. Twelve of 52 neuroblast clones exhibited obvious dorsal lobe axon overextension not seen in control flies. These include overextension of thin axon bundles near the tip of dorsal lobe similar to those seen in Drok2 clones, or overextension of a large portion of the dorsal axons similar to those seen in MB neurons overexpressing p190. This experiment indicates that integrins are essential negative regulators of axon extension in MB neurons (Billuart, 2001).
Billuart, P., Winter, C. G., Maresh, A., Zhao, X. and Luo, L. (2001). Regulating axon branch stability. the role of p190 RhoGAP in repressing a retraction signaling pathway. Cell 107(2): 195-207. 11672527
Brouns, M. R., et al. (2000). The adhesion signaling molecule p190 RhoGAP is required for morphogenetic processes in neural development. Development 127: 4891-4903. 11044403
Brouns, M. R., Matheson, S. F. and Settleman, J. (2001). p190 RhoGAP is the principal Src substrate in brain and regulates axon outgrowth, guidance and fasciculation. Nat. Cell Biol. 3: 361-367. 11283609
Burbelo, P. D., et al. (1995). p190-B, a new member of the Rho GAP family, and Rho are induced to cluster after integrin cross-linking. J. Biol. Chem. 270(52): 30919-30926.
Bustos, R. I., Forget, M. A., Settleman, J. E. and Hansen, S. H. (2008). Coordination of Rho and Rac GTPase function via p190B RhoGAP. Curr. Biol. 18(20): 1606-11. PubMed Citation: 18948007
Chang, J., Gill, S., Settleman, J. and Parsons, S. (1995). c-Src regulates the simultaneous rearrangement of actin cytoskeleton, p190RhoGAP following epidermal growth factor stimulation. J. Cell Biol. 130: 355-368. 7542246
de Bettignies, G., et al. (2001). Overactivation of the Protein kinase C-signaling pathway suppresses the defects of cells lacking the Rho3/Rho4-GAP Rgd1p in Saccharomyces cerevisiae. Genetics 159: 1435-1448. 11779787
Dib, K., Melander, F. and Andersson, T. (2001). Role of p190RhoGAP in beta 2 integrin regulation of RhoA in human neutrophils. J. Immunol. 166(10): 6311-22. 11342655
Diogon, M., et al. (2007). The RhoGAP RGA-2 and LET-502/ROCK achieve a balance of actomyosin-dependent forces in C. elegans epidermis to control morphogenesis. Development 134: 2469-2479. Medline abstract: 17537791
Ellis, C., Moran, M., McCormick, F. and Pawson, T. (1990). Phosphorylation of GAP and GAP-associated proteins by transforming and mitogenic tyrosine kinases. Nature 343: 377-381. 1689011
Fincham, V. J., Chudleigh, A. and Frame, M. C. (1999). Regulation of p190 Rho-GAP by v-Src is linked to cytoskeletal disruption during transformation. J. Cell Sci. 112:947-956. 10036244
Haskell, M. D., et al. (2001). Phosphorylation of p190 on Tyr 1105 by c-Src is necessary but not sufficient for EGF-induced actin disassebly in C3H10T1/2 fibroblasts. J. Cell Sci. 114: 1699-1708. 11309200
Hernández, S. E., Settleman, J. and Koleske, A. J. (2004). Adhesion-dependent regulation of p190RhoGAP in the developing brain by the Abl-related gene tyrosine kinase. Curr. Biol. 14: 691-696. 15084284
Lamprecht, R., Farb, C. R. and LeDoux, J. E. (2002). Fear memory formation involves p190 RhoGAP and ROCK proteins through a GRB2-mediated complex. Neuron 36: 727-738. 12441060
Lee, T., et al. (2000). Essential roles of Drosophila RhoA in the regulation of neuroblast proliferation and dendritic but not axonal morphogenesis. Neuron 25: 307-316. PubMed Citation: 10719887
Nakahara, H., et al. (1998). Activation of beta1 integrin signaling stimulates tyrosine phosphorylation of p190RhoGAP and membrane-protrusive activities at invadopodia. J. Biol. Chem. 273(1): 9-12. PubMed Citation: 9417037
Nakayama, A. Y., Harms, M. B. and Luo, L. (2000). Small GTPases Rac and Rho in the maintenance of dendritic spines and branches in hippocampal pyramidal neurons. J. Neurosci. 20: 5329-5338. 10884317
Ridley, A. J., et al. (1993). rho family GTPase activating proteins p190, bcr and rhoGAP show distinct specificities in vitro and in vivo. EMBO J. 12: 5151-5160. 8262058
Roof, R. W., et al. (1998). Phosphotyrosine (p-Tyr)-dependent and independent mechanisms of p190 RhoGAP-p120 RasGAP interactions: Tyr 1105 of p190, a substrate for c-Src, is the sole p-Tyr mediator of complex formation. Mol. Cell Biol. 18: 7052-7063. 9819392
Roof, R. W., Dukes, B. D., Chang, J.-H. and Parsons, S. J. (2000). Phosphorylation of the p190 RhoGAP N-terminal domain by c-Src results in a loss of GTP binding activity. FEBS Lett. 472: 117-121. 10781817
Schmutz, C., Stevens, J. and Spang, A. (2007). Functions of the novel RhoGAP proteins RGA-3 and RGA-4 in the germ line and in the early embryo of C. elegans. Development 134(19): 3495-505. PubMed citation: 17728351
Schonegg, S., Constantinescu, A. T., Hoege, C. and Hyman, A. A. (2007). The Rho GTPase-activating proteins RGA-3 and RGA-4 are required to set the initial size of PAR domains in Caenorhabditis elegans one-cell embryos. Proc. Natl. Acad. Sci. 104(38): 14976-81. Medline abstract: 17848508
Settleman, J., Narasimhan, V., Foster, L. and Weinberg, R. (1992). Molecular cloning of cDNAs encoding the GAP-associated protein P190: Implications for a signaling pathway from Ras to the nucleus. Cell 69: 539-549. 1581965
Sharma, S. V. (1998). Rapid recruitment of p120RasGAP and its associated protein, p190RhoGAP, to the cytoskeleton during integrin mediated cell-substrate interaction. Oncogene 17: 271-281. 9690509
Sordella, R., et al. (2002). Modulation of CREB activity by the Rho GTPase regulates cell and organism size during mouse embryonic development. Dev. Cell 2: 553-565. 12015964
Sordella, R., et al. (2003). Modulation of Rho GTPase signaling regulates a switch between adipogenesis and myogenesis. Cell 113: 147-158. 12705864
Sotillos, S. and Campuzano, S. (2000). DRacGAP, a novel Drosophila gene, inhibits EGFR/Ras signalling in the developing imaginal wing disc. Development 127: 5427-5438. 11076763
Tatsis, N., Lannigan, D. and Macara, I. (1998). The function of the p190 Rho GTPase-activating protein is controlled by its N-terminal GTP binding domain. J. Biol. Chem. 273: 34631-34638. 9852136
Winter, C. G., et al. (2001). Drosophila Rho-associated kinase (Drok) links Frizzled-mediated planar cell polarity signaling to the actin cytoskeleton. Cell 105: 81-91. 11301004
Wolf, R. M., Wilkes, J. J., Chao, M. V. and Resh, M. D. (2001). Tyrosine phosphorylation of p190 RHOGAP by Fyn regulates oligodendrocyte differentiation. J Neurobiol. 49(1): 62-78. 11536198
Zhang, H. and Macara, I. G. (2008). The PAR-6 polarity protein regulates dendritic spine morphogenesis through p190 RhoGAP and the Rho GTPase. Dev. Cell 14(2): 216-26. PubMed Citation: 18267090
date revised: 25 February 2009
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