RhoA and rho kinase regulate the epithelial Na+/H+ exchanger NHE3. Role of myosin light chain phosphorylation.
The activity of the Na(+)/H(+) exchanger NHE3 isoform, which is found primarily in epithelial cells, is sensitive to the state of actin polymerization. Actin assembly, in turn, is controlled by members of the small GTPase Rho family, namely Rac1, Cdc42, and RhoA. We therefore investigated the possible role of these GTPases in modulating NHE3 activity. Cells stably expressing NHE3 were transiently transfected with inhibitory forms of Rac1, Cdc42, or RhoA and transport activity was assessed using microfluorimetry. NHE3 activity was not adversely affected by either dominant-negative Rac1 or Cdc42. By contrast, the inhibitory form of RhoA greatly depressed NHE3 activity, without noticeably altering its subcellular distribution. NHE3 activity was equally reduced by inhibiting p160 Rho-associated kinase I (ROK), a downstream effector of RhoA, with the selective antagonist Y-27632 and a dominant-negative form of ROK. Furthermore, inhibition of ROK reduced the phosphorylation of myosin light chain. A comparable net dephosphorylation was achieved by the myosin light chain kinase inhibitor ML9, which similarly inhibited NHE3. These data suggest that optimal NHE3 activity requires a functional RhoA-ROK signaling pathway which acts, at least partly, by controlling the phosphorylation of myosin light chain and, ultimately, the organization of the actin cytoskeleton.
Szászi
K
K
Cell Biology Programme, The Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada.
Kurashima
K
K
Kapus
A
A
Paulsen
A
A
Kaibuchi
K
K
Grinstein
S
S
Orlowski
J
J
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D047908:Intracellular Signaling Peptides and Proteins; D018994:Myosin Light Chains / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers / Q000502:physiology*; D014162:Transfection; D020764:cdc42 GTP-Binding Protein / Q000502:physiology; D020830:rac1 GTP-Binding Protein / Q000502:physiology; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein / Q000502:physiology*
D047908:Intracellular Signaling Peptides and Proteins; D018994:Myosin Light Chains; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D020764:cdc42 GTP-Binding Protein; D020830:rac1 GTP-Binding Protein; D020742:rhoA GTP-Binding Protein