The GTP binding proteins Gem and Rad are negative regulators of the Rho–Rho kinase pathway

Abstract

The cytoskeletal changes that alter cellular morphogenesis and motility depend upon a complex interplay among molecules that regulate actin, myosin, and other cytoskeletal components. The Rho family of GTP binding proteins are important upstream mediators of cytoskeletal organization. Gem and Rad are members of another family of small GTP binding proteins (the Rad, Gem, and Kir family) for which biochemical functions have been mostly unknown. Here we show that Gem and Rad interface with the Rho pathway through association with the Rho effectors, Rho kinase (ROK) α and β. Gem binds ROKβ independently of RhoA in the ROKβ coiled-coil region adjacent to the Rho binding domain. Expression of Gem inhibited ROKβ-mediated phosphorylation of myosin light chain and myosin phosphatase, but not LIM kinase, suggesting that Gem acts by modifying the substrate specificity of ROKβ. Gem or Rad expression led to cell flattening and neurite extension in N1E-115 neuroblastoma cells. In interference assays, Gem opposed ROKβ- and Rad opposed ROKα-mediated cell rounding and neurite retraction. Gem did not oppose cell rounding initiated by ROKβ containing a deletion of the Gem binding region, demonstrating that Gem binding to ROKβ is required for the effects observed. In epithelial or fibroblastic cells, Gem or Rad expression resulted in stress fiber and focal adhesion disassembly. In addition, Gem reverted the anchorage-independent growth and invasiveness of Dbl-transformed fibroblasts. These results identify physiological roles for Gem and Rad in cytoskeletal regulation mediated by ROK.

The RGK family of GTP-binding proteins: Regulators of voltage-dependent calcium channels and cytoskeleton remodeling

“…ROK is a central effector for Rho GTPases [62][63][64][65] and the direct interaction of Gem with Rho kinase-β [50] inhibits ROKβ-mediated phosphorylation of both the myosin light chain (MLC) and myosin-binding subunit (MBS) of myosin light chain phosphatase [50]. In a similar fashion, Rad has been shown to associate with the ROK-α isoform [50] (Fig. 2).…”

“…Finally, it is important to note that both Rem and Rem2 have been reported to induce cytoskeletal reorganization [41,43,47,58]. However, it remains unclear whether Rem or Rem2 can activate Gmip and neither protein associates with ROK [50], suggesting that an additional regulatory mechanism may remain to be defined. Thus, while RGK GTPases regulate both cell morphology and migration, questions remain concerning both the molecular mechanism(s) and cellular stimuli that control these pathways.…”

“…RGK proteins do not contain canonical lipid modification motifs [48], though there is enrichment of these proteins at the plasma membrane, and individual proteins have been shown to be localized to the cytosol, nucleus, and with both the actin and microtubule networks [4,5,18,41,43,44,[46][47][48][49][50][51][52][53][54]. The carboxyl terminus is well conserved (Fig.…”

“…More importantly, overexpression of Gem or Rad has been shown to antagonize Rho kinase (ROK) induced neurite retraction and cause morphological differentiation in neuroblastoma cells [26,42,50], stress fiber disassembly and focal adhesion dissolution in fibroblasts and epithelial cells [42,50], while expression of a putative dominant-negative Rad mutant inhibits these processes in vascular smooth muscle cells [21]. ROK is a central effector for Rho GTPases [62][63][64][65] and the direct interaction of Gem with Rho kinase-β [50] inhibits ROKβ-mediated phosphorylation of both the myosin light chain (MLC) and myosin-binding subunit (MBS) of myosin light chain phosphatase [50]. In a similar fashion, Rad has been shown to associate with the ROK-α isoform [50] (Fig.…”

Rho kinase in the regulation of cell death and survival

“…The small GTP-binding protein RhoE interacts with the N-terminal region of ROCK1 (amino acids 1-420) and prevents Rho binding to RBD [101,112]. Other negative regulators have been found to bind to and inhibit ROCK activity, such as the small GTP-binding proteins, Gem and Rad [145], Raf-1 [107], p21(Cip1/WAF1) [81] and Aurora-A/serine/threonine kinase 15 (STK15) [25]. However, their mechanisms of action are not defined.…”