Phosphorylation-dependent activation of the Ras-GRF/CDC25Mm exchange factor by muscarinic receptors and G-protein βγ subunits

  title={Phosphorylation-dependent activation of the Ras-GRF/CDC25Mm exchange factor by muscarinic receptors and G-protein $\beta$$\gamma$ subunits},
  author={Raymond R. Mattingly and Ian G. Macara},
MUSCARINIC receptors activate Ras through a pathway distinct1,2 from that mediated through translocation of the exchange factor mSos1 by receptor tyrosine kinases3,4. Here we report that muscarinic receptors can activate another Ras exchange factor, CDC25Mm, or p140Ras-GRF (refs 5,6). In NIH-3T3 cells expressing subtype 1 human muscarinic receptors (hml), the agonist carbachol selectively increased the specific activity and phosphorylation state of epitope-tagged Ras-GRF. This stimulation was… 

Phosphorylation of Serine 916 of Ras-GRF1 Contributes to the Activation of Exchange Factor Activity by Muscarinic Receptors*

  • R. Mattingly
  • Biology, Chemistry
    The Journal of Biological Chemistry
  • 1999
Serine 916 was a substrate for protein kinase A both in vivo and in vitro, suggesting a novel link between the cAMP and Ras signaling systems and phosphorylation at residue 916 is necessary for full activation of Ras-GRF1.

Phosphorylation of the Ras-GRF1 Exchange Factor at Ser916/898 Reveals Activation of Ras Signaling in the Cerebral Cortex*

It is demonstrated here that Ras-GRF1 is highly expressed in rat brain compared with the Sos exchange factor and that there is an increase in incorporation of 32P into Ser898 of brain Ras- GRF1 following activation of protein kinase A.

Stimulation of Ras Guanine Nucleotide Exchange Activity of Ras-GRF1/CDC25Mm upon Tyrosine Phosphorylation by the Cdc42-regulated Kinase ACK1*

It is shown here that activated ACK1, a nonreceptor tyrosine kinase that belongs to the focal adhesion kinase family, causes tyosine phosphorylation of Ras-GRF1 and suggest a signaling cascade consisting of Cdc42, ACK 1, Ras- GRF1, and Ras in neuronal cells.

Induction of Rac-Guanine Nucleotide Exchange Activity of Ras-GRF1/CDC25Mm following Phosphorylation by the Nonreceptor Tyrosine Kinase Src*

It is shown that the nonreceptor tyrosine kinase Src phosphorylates Ras-GRF1, thereby inducing Rac-GEF activity, which may imply the involvement of Src downstream of Gβγ to regulate Ras- GRF1.

G protein βγ subunit-dependent Rac-guanine nucleotide exchange activity of Ras-GRF1/CDC25Mm

It is shown that Ras-GRF1 exhibits Rac1-specific GEF activity when recovered from cells overexpressing G protein βγ subunits (Gβγ), suggesting a role of Ras- GRF1 for regulating Rac-dependent as well as Ras-dependent signaling pathways, particularly in the brain functions.

Maintenance of Cdc42 GDP-bound State by Rho-GDI Inhibits MAP Kinase Activation by the Exchange Factor Ras-GRF

The results suggest that the Cdc42 mechanism of action may not be solely restricted to activation of downstream signaling cascades when GTP-loaded, and the GDP-bound form may be acting as an inhibitory molecule down-modulating parallel signaling routes such as the Ras/MAPK pathway.

Sites of Phosphorylation by Protein Kinase A in CDC25Mm/GRF1, a Guanine Nucleotide Exchange Factor for Ras*

The involvement of cAMP-dependent protein kinase (PKA) in the phosphorylation of CDC25Mm in fibroblasts overexpressing this RasGEF as well as in mouse brain synaptosomal membranes is demonstrated and multiple PKA-dependent phosphorylated sites suggest a complex regulatory picture of this Ras GEF.

Ras-Specific Exchange Factor GRF: Oligomerization through Its Dbl Homology Domain and Calcium-Dependent Activation of Raf

It is concluded that GRF 1 and GRF2 can form homo- and hetero-oligomers via their DH domains, that mutational inactivation of oligomer formation by GRF1 is associated with impaired biological and signaling activities, and that in 293T cellsGRF1 mediates at least two pathways for Raf activation.

Calcium and calmodulin are essential for Ras-GRF1-mediated activation of the Ras pathway by lysophosphatidic acid.

Calmodulin and cytosolic free calcium are essential for Ras and MAPK activation induced by LPA and mediated by Ras-GRF1, as shown by the finding that BAPTA-AM, an intracellular calcium chelator, and calmodulin inhibitors completely abolished this effect.



Muscarinic receptors transform NIH 3T3 cells through a Ras-dependent signalling pathway inhibited by the Ras-GTPase-activating protein SH3 domain.

Through cotransfection experiments and measurement of the activation state of native and epitope-tagged Ras proteins, the contributions of Ras and Ras GTPase-activating protein (Ras-GAP) to muscarinic receptor-dependent transformation were defined.

Differential response of the Ras exchange factor, Ras-GRF to tyrosine kinase and G protein mediated signals.

The results suggest that Ras-GRF has the capacity to mediate Ras activation initiated by signals using heterotrimeric G proteins.

Regulated and constitutive activity by CDC25Mm (GRF), a Ras-specific exchange factor

It is concluded that each type of CDC25Mm induces cell transformation via the ability of its C terminus to stimulate guanine nucleotide exchange on Ras, the presence of N-terminal sequences is associated with a serum-dependent change in GTP, and the level of GTP increased in NIH 3T3 cells and in cells expressing the type IV protein.

Molecular cloning of cDNAs encoding a guanine-nucleotide-releasing factor for Ras p21

The cloning of complementary DNAs from a rat brain library that encode a ∼140 GRF for Ras p21 (p140Ras-GRF) is reported, which accelerated the release of GDP from RasH and RasN p21 in vitro, but not from the related RalA, or CDC42Hs GTP-binding proteins.

Distinct Pathways of G- and G-mediated Mitogen-activated Protein Kinase Activation (*)

The role of Gβ in Gi- and Gq-coupled receptor-mediated PI hydrolysis and MAPK activation is defined and direct stimulation with Gβ resulted in MAPKactivation that was sensitive to inhibition by expression of βARKct, RasN17, or NΔRaf or by PTK inhibitors, but insensitive to PKC depletion.

Regulation of Epidermal Growth Factor Receptor Signaling by Phosphorylation of the Ras Exchange Factor hSOS1 (*)

Phosphorylation of hSOS1, by affecting its interaction with the hEGFR or Shc, down-regulates signal transduction from the h EGFR to the Ras pathway.

Muscarinic acetylcholine receptor subtypes as agonist-dependent oncogenes.

It is demonstrated that mAChRs linked to phosphatidylinositol hydrolysis can act as conditional oncogenes when expressed in cells capable of proliferation.