The RAS effector RIN1 directly competes with RAF and is regulated by 14-3-3 proteins.
Activation of RAS proteins can lead to multiple outcomes by virtue of regulated signal traffic through alternate effector pathways. We demonstrate that the RAS effector protein RIN1 binds to activated RAS with an affinity (K(d), 22 nM) similar to that observed for RAF1. At concentrations close to their equilibrium dissociation constant values, RIN1 and RAF1 compete directly for RAS binding. RIN1 was also observed to inhibit cellular transformation by activated mutant RAS. This distinguishes RIN1 from other RAS effectors, which are transformation enhancing. Blockade of transformation was mediated by the RAS binding domain but required membrane localization. RIN1 recognizes endogenous RAS following transient activation by epidermal growth factor, and a portion of RIN1 fractionates to the cell membrane in a manner consistent with a reversible interaction. RIN1 also binds to 14-3-3 proteins through a sequence including serine 351. Mutation of this residue abolished the 14-3-3 binding capacity of RIN1 and led to more efficient blockade of RAS-mediated transformation. The mutant protein, RIN1(S351A), showed a shift in localization to the plasma membrane. Serine 351 is a substrate for protein kinase D (PKD [also known as PKCmu]) in vitro and in vivo. These data suggest that the normal localization and function of RIN1, as well as its ability to compete with RAF, are regulated in part by 14-3-3 binding, which in turn is controlled by PKD phosphorylation.
Wang
Ying
Y
Department of Biological Chemistry, University of California, Los Angeles, Los Angeles, California 90095, USA.
Waldron
Richard T
RT
Dhaka
Ajay
A
Patel
Apoor
A
Riley
Maggie M
MM
Rozengurt
Enrique
E
Colicelli
John
J
D048948:14-3-3 Proteins; D016475:3T3 Cells; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D051379:Mice; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D015398:Signal Transduction; D014170:Transformation, Genetic; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism*; D020691:rab GTP-Binding Proteins; D018631:ras Proteins / Q000378:metabolism*
D048948:14-3-3 Proteins; D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; C092117:RIN1 protein, human; D011994:Recombinant Proteins; C490451:Rin1 protein, mouse; C490450:Rin1 protein, rat; D012694:Serine; D014446:Tyrosine 3-Monooxygenase; C089212:protein kinase D; D019908:Proto-Oncogene Proteins c-raf; D011493:Protein Kinase C; D020691:rab GTP-Binding Proteins; D018631:ras Proteins