Rho family members: Activators of MAP kinase cascades

Low molecularweight GTP-binding proteins are molecular switches that control a diverse array of biological processes (Bourne et al., 1991). When bound to GTP, these proteins transduce signals to effector proteins. When bound to GDP, these proteins are in an inactive state. The cycling between the active, GTP-bound state and the inactive, GDP-bound state is controlled by positive and negative modulators that directly contact the GTP-binding protein. Positive modulators, guanine nucleotide dissociation stimulators (GDSs), catalyze the dissociation of GDP. Subsequently, GTP binds, and the protein is active. Negative modulators, GTPase-activating proteins (GAPS), stimulate the intrinsic GTP hydrolytic activity of the GTPbinding proteins and, hence, the formation of the inactive, GDP-bound state. Approximately 50 low molecular weight GTPases have been described (Chardin, 1991). The family members can be subdivided, on the basis of sequence homology, into five classes: Ras, Rab, Arf, Ran, and Rho. Ras family members (H-Ras, K-Ras, N-Ras, R-Ras, TC21, RaplAl Rap1 B, and RapPAIRapPB) play salient roles in cell growth and development. The members of the Rab/Arf subfamilies monitor and direct the movements of vesicles within the cell. Ran is required for nuclear protein import. And, finally, the Rho family members (Cdc42/G25K, Racl, Rac2, RhoA, RhoB, and RhoC) play dynamic roles in the regulation of the actin cytoskeleton. Regulated changes in the actin cytoskeleton are required for cytokinesis, for cell motility, for vesicle trafficking, for pinocytosis, and, in the yeast Saccharomyces cerevisiae, for bud site selection and for polarized cell growth. Each of the small GTPases of the Rho subfamily participates in distinct patterns of actin reorganization. Rat is required for membrane ruffling, whereas Rho is required for the formation of stress fibers (Ridley and Hall, 1992; Ridley et al., 1992). Cdc42 was first identified in the yeast S. cerevisiae, where it is required for polarized cell growth (Adamset al., 1990); subsequently, a homolog of this yeast protein, Cdc42Hs (G25K), was identified from mammalian cells, where it regulates the formation of actin-containing microspikes, called filopodia (Kozma et al., 1995; Nobes and Hall, 1995). The observation that chronic activation of Cdc42, Rat, and Rho by deregulated exchange factors induces malignant transformation as well as morphological changes (Khosravifar et al., 1994; Michiels et al., 1995) has suggested that these GTPases may regulate nuclear as well as cytoplasmic effects. Three recent papers in Cell (Coso et al., 1995; Minden et al., 1995; Hill et al., 1995) describe a link from these GTP-binding proteins to the nucleus and, for Cdc42 and Rat, to a mitogen-activating protein kinase (MAPK) cascade. Coso et al. (1995) and Minden et al. (1995) made extensive use of transient transfection assays with an epitopetagged cJun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK) cDNA clone as reporter. Protein kinase assays of the recovered tagged JNK were used to monitor activity of the MAPK pathway that leads to the JNKs (in order, MAP or extracellular-regulated kinase [ERK] kinase 1 [MEKKl], SAP or ERK kinase [SEKIIMAPK kinase 4 [MKK4], and JNK; Figure 1). By cotransfecting various mutants of Cdc42, Racl, and RhoA that lock the small GTPase into a constitutively active or inactive state, Coso et al. and Minden et al. were able to show that JNK activation is dependent on Cdc42 and Racl , but not RhoA. In addition, transfection of a RhoGAP, which inactivates all Rho family members, reduces activation of JNK by cytokines, and transfection of Dbl or Ost, GDSs for Cdc42 and also RhoA, efficiently activates JNK. The Dbl-induced activation of JNK was abolished upon cotransfection of JNK with an N-terminal region of the serinelthreonine kinase PAK85 (Manser et al., 1994; Martin et al., 1995) which includes a Cdc42IRacbinding domain and may be expected to block signaling from Dbl through Cdc42 by titrating the GTPase. This result indicates that the Dbl activation of JNK is mediated through its activation of Cdc42, not RhoA. Minden et al. also detected activation of Jun (a JNK substrate) in cells expressing activated Rat and showed that Rat activation of JNK is blocked by kinasedefective mutants of either SEWMKK4 or MEKKl, which are expected to sequester their respective activators. These results suggest that Cdc42 and Racl can activate