The GTPase-deficient Rnd proteins are stabilized by their effectors.
Rnd proteins are Rho family GTP-binding proteins with cellular functions that antagonize RhoA signaling. We recently described a new Rnd3 effector Syx, also named PLEKHG5, that interacts with Rnds via a Raf1-like "Ras-binding domain." Syx is a multidomain RhoGEF that participates in early zebrafish development. Here we demonstrated that Rnd1, Rnd2, and Rnd3 stability is acutely dependent on interaction with their effectors such as Syx or p190 RhoGAP. Although Rnd3 turnover is blocked by treatment of cells with MG132, we provide evidence that such turnover is mediated indirectly by effects on the Rnd3 effectors, rather than on Rnd3 itself, which is not significantly ubiquitinated. The minimal regions of Syx and p190 RhoGAP that bind Rnd3 are not sequence-related but have similar effects. We have identified features that allow for Rnd3 turnover including a conserved Lys-45 close to the switch I region and the C-terminal membrane-binding domain of Rnd3, which cannot be substituted by the equivalent Cdc42 CAAX sequence. By contrast, an effector binding-defective mutant of Rnd3 when overexpressed undergoes turnover at normal rates. Interestingly the activity of the RhoA-regulated kinase ROCK stimulates Rnd3 turnover. This study suggests that Rnd proteins are regulated through feedback mechanisms in cells where the level of effectors and RhoA activity influence the stability of Rnd proteins. This effector feedback behavior is analogous to the ability of ACK1 and PAK1 to prolong the lifetime of the active GTP-bound state of Cdc42 and Rac1.
Rho GTPases Signaling Group, Institute of Medical Biology, 8A Biomedical Grove, 06-06 Immunos Building 138648, Singapore.
Liuh Ling
LL
Goh
Ed
E
Manser
D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability / Q000187:drug effects / Q000502:physiology; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D007976:Leupeptins / Q000494:pharmacology; D009154:Mutation; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D015027:Zebrafish; D020764:cdc42 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*; D054462:p21-Activated Kinases / Q000235:genetics / Q000378:metabolism*; D020830:rac1 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*; D020741:rho GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D054460:rho-Associated Kinases / Q000378:metabolism
C074560:ARHGAP35 protein, human; D015853:Cysteine Proteinase Inhibitors; D020662:Guanine Nucleotide Exchange Factors; D007976:Leupeptins; C107953:RAC1 protein, human; D012097:Repressor Proteins; D011505:Protein-Tyrosine Kinases; C403262:TNK2 protein, human; C515903:PAK1 protein, human; D054462:p21-Activated Kinases; D054460:rho-Associated Kinases; D020764:cdc42 GTP-Binding Protein; D020830:rac1 GTP-Binding Protein; D020741:rho GTP-Binding Proteins; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde