Effect of Phosphorylation on Activities of Rap1A to Interact with Raf-1 and to Suppress Ras-dependent Raf-1 Activation*

Rap1A is phosphorylated by cAMP-dependent protein kinase (PKA), and this phosphorylation has been shown to modulate its interaction with other proteins. However, it is not known whether Rap1A phosphorylation is involved in regulation of its cellular functions, including suppression of Ras-dependent Raf-1 activation. We have previously shown that this suppressive activity of Rap1A is attributable to its greatly enhanced ability to bind to the cysteine-rich region (CRR, residues 152–184) of Raf-1 compared with that of Ras. Here, we show that phosphorylation of Rap1A by PKA abolished its binding activity to CRR. Furthermore, a mutant Rap1A(S180E), whose sole PKA phosphorylation residue, Ser-180, was substituted by an acidic residue, Glu, to mimic its phosphorylated form, failed to suppress Ras-dependent Raf-1 activation in COS-7 cells. These results indicate that the CRR binding activity and the Ras-suppressive function of Rap1A can be modulated through phosphorylation and suggest that Rap1A may function as a PKA-dependent regulator of Raf-1 activation, not merely as a suppressor.

[1]  T. Kataoka,et al.  Identification of PLC210, a Caenorhabditis elegansPhospholipase C, as a Putative Effector of Ras* , 1998, The Journal of Biological Chemistry.

[2]  T. Kataoka,et al.  Membrane recruitment of Raf-1 is not the only function of Ras in Raf-1 activation , 1997, Oncogene.

[3]  Sandrine Roy,et al.  Activity of Plasma Membrane-recruited Raf-1 Is Regulated by Ras via the Raf Zinc Finger* , 1997, The Journal of Biological Chemistry.

[4]  S. Yokoyama,et al.  Coassociation of Rap1A and Ha-Ras with Raf-1 N-terminal Region Interferes with Ras-dependent Activation of Raf-1* , 1997, The Journal of Biological Chemistry.

[5]  H. Yao,et al.  cAMP Activates MAP Kinase and Elk-1 through a B-Raf- and Rap1-Dependent Pathway , 1997, Cell.

[6]  D. Morrison,et al.  The complexity of Raf-1 regulation. , 1997, Current opinion in cell biology.

[7]  J. Avruch,et al.  An intact Raf zinc finger is required for optimal binding to processed Ras and for ras-dependent Raf activation in situ , 1997, Molecular and cellular biology.

[8]  W. Kolch,et al.  Negative regulation of Raf-1 by phosphorylation of serine 621 , 1996, Molecular and cellular biology.

[9]  S. Yokoyama,et al.  Cysteine-rich Region of Raf-1 Interacts with Activator Domain of Post-translationally Modified Ha-Ras (*) , 1995, The Journal of Biological Chemistry.

[10]  A. Wittinghofer,et al.  The 2.2 Å crystal structure of the Ras-binding domain of the serine/threonine kinase c-Raf1 in complex with RaplA and a GTP analogue , 1995, Nature.

[11]  C. Der,et al.  Two Distinct Raf Domains Mediate Interaction with Ras (*) , 1995, The Journal of Biological Chemistry.

[12]  J. Bos,et al.  Regulation of Ras-mediated signalling: more than one way to skin a cat. , 1995, Trends in biochemical sciences.

[13]  R. Bell,et al.  Identification of discrete segments of human Raf-1 kinase critical for high affinity binding to Ha-Ras. , 1994, The Journal of biological chemistry.

[14]  S. Fields,et al.  Activated Ras interacts with the Ral guanine nucleotide dissociation stimulator. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[15]  J. Troppmair,et al.  The ins and outs of Raf kinases. , 1994, Trends in biochemical sciences.

[16]  S. Demo,et al.  ralGDS family members interact with the effector loop of ras p21 , 1994, Molecular and cellular biology.

[17]  W. Xie,et al.  The cysteine-rich region of raf-1 kinase contains zinc, translocates to liposomes, and is adjacent to a segment that binds GTP-ras. , 1994, The Journal of biological chemistry.

[18]  S. Cook,et al.  RapV12 antagonizes Ras‐dependent activation of ERK1 and ERK2 by LPA and EGF in Rat‐1 fibroblasts. , 1993, The EMBO journal.

[19]  M. Noda.  Structures and functions of the K rev-1 transformation suppressor gene and its relatives. , 1993, Biochimica et biophysica acta.

[20]  T. Kataoka,et al.  The effect of posttranslational modifications on the interaction of Ras2 with adenylyl cyclase , 1993, Science.

[21]  R. Weinberg,et al.  Characterization of a guanine nucleotide dissociation stimulator for a ras‐related GTPase. , 1993, The EMBO journal.

[22]  G. Bokoch.  Biology of the Rap proteins, members of the ras superfamily of GTP-binding proteins. , 1993, The Biochemical journal.

[23]  A. Jesaitis,et al.  Inhibition of Rap1A binding to cytochrome b558 of NADPH oxidase by phosphorylation of Rap1A. , 1991, Science.

[24]  C. Der,et al.  Rap1A is a substrate for cyclic AMP-dependent protein kinase in human neutrophils. , 1991, Journal of immunology.

[25]  J. de Gunzburg,et al.  The cAMP-dependent protein kinase phosphorylates the rap1 protein in vitro as well as in intact fibroblasts, but not the closely related rap2 protein. , 1991, Biochemical and biophysical research communications.

[26]  S. Orkin,et al.  Association of a Ras-related protein with cytochrome b of human neutrophils , 1989, Nature.

[27]  H. Kitayama,et al.  A ras-related gene with transformation suppressor activity , 1989, Cell.

[28]  A. Smith,et al.  A potent synthetic peptide inhibitor of the cAMP-dependent protein kinase. , 1986, The Journal of biological chemistry.

[29]  K. Mullis,et al.  Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. , 1985, Science.