Phosphorylation of the Ras nucleotide exchange factor son of sevenless by mitogen-activated protein kinase
UMass Chan AffiliationsProgram in Molecular Medicine
Document TypeJournal Article
Calcium-Calmodulin-Dependent Protein Kinases
Guanine Nucleotide Exchange Factors
Hemagglutinin Glycoproteins, Influenza Virus
Molecular Sequence Data
Polymerase Chain Reaction
Recombinant Fusion Proteins
Son of Sevenless Proteins
ras Guanine Nucleotide Exchange Factors
Medicine and Health Sciences
MetadataShow full item record
AbstractSon of sevenless-1 and -2 (Sos-1 and -2) are guanosine nucleotide exchange factors implicated in the activation of Ras by both the insulin and epidermal growth factor signal transduction pathways. Ras appears to function by initiating the activation of cellular protein kinases including mitogen-activated protein (MAP) kinases. Sos proteins contain numerous sequences in their carboxyl-terminal regions which correspond to consensus sites for MAP kinase phosphorylation. To examine whether these sites are substrates for MAP kinases, the cDNA encoding Drosophila Sos (dSos) was tagged with sequences encoding the major antigenic epitope of the influenza virus hemagglutinin (HA) to create a dSosHA fusion construct. dSosHA was transiently expressed in COS-1 cells and immunoprecipitated with anti-HA antibodies. When immune complexes were incubated with purified MAP kinase and [gamma-32P]ATP, a phosphorylated band of 180 kDa was observed when analyzed by SDS-polyacrylamide gel electrophoresis. This band was not present in immunoprecipitations from cells transfected with vector alone. No phosphorylation of the 180 kDa band was seen when immunoprecipitates were incubated with [gamma-32P]ATP in the absence of MAP kinase. Two dimensional analysis of tryptic peptides from dSosHA phosphorylated by MAP kinase in vitro revealed two major phosphorylated species that were also found in dSosHA isolated from COS-1 cells labeled with 32Pi. These results are consistent with the hypothesis that a feedback loop exists wherein growth factor-activated MAP kinases phosphorylate and regulate Sos proteins.
SourceJ Biol Chem. 1994 Feb 18;269(7):4717-20.
Permanent Link to this Itemhttp://hdl.handle.net/20.500.14038/42480
Related ResourcesLink to Article in PubMed
Showing items related by title, author, creator and subject.
Dynamic Regulation at the Neuronal Plasma Membrane: Novel Endocytic Mechanisms Control Anesthetic-Activated Potassium Channels and Amphetamine-Sensitive Dopamine Transporters: A DissertationGabriel, Luke R. (2013-06-13)Endocytic trafficking dynamically regulates neuronal plasma membrane protein presentation and activity, and plays a central role in excitability and plasticity. Over the course of my dissertation research I investigated endocytic mechanisms regulating two neuronal membrane proteins: the anesthetic-activated potassium leak channel, KCNK3, as well as the psychostimulant-sensitive dopamine transporter (DAT). My results indicate that KCNK3 internalizes in response to Protein Kinase C (PKC) activation, using a novel pathway that requires the phosphoserine binding protein, 14-3-3β, and demonstrates for the first time regulated KCNK3 channel trafficking in neurons. Additionally, PKC-mediated KCNK3 trafficking requires a non-canonical endocytic motif, which is shared exclusively between KCNK3 and sodium-dependent neurotransmitter transporters, such as DAT. DAT trafficking studies in intact ex vivo adult striatal slices indicate that DAT endocytic trafficking has both dynamin-dependent and –independent components. Moreover, DAT segregates into two populations at the neuronal plasma membrane: trafficking-competent and -incompetent. Taken together, these results demonstrate that novel, non-classical endocytic mechanisms dynamically control the plasma membrane presentation of these two important neuronal proteins.
Selective interaction of JNK protein kinase isoforms with transcription factorsGupta, Shashi; Barrett, Tamera; Whitmarsh, Alan J.; Cavanagh, Julie; Sluss, Hayla Karen; Derijard, Benoit; Davis, Roger J. (1996-06-03)The JNK protein kinase is a member of the MAP kinase group that is activated in response to dual phosphorylation on threonine and tyrosine. Ten JNK isoforms were identified in human brain by molecular cloning. These protein kinases correspond to alternatively spliced isoforms derived from the JNK1, JNK2 and JNK3 genes. The protein kinase activity of these JNK isoforms was measured using the transcription factors ATF2, Elk-1 and members of the Jun family as substrates. Treatment of cells with interleukin-1 (IL-1) caused activation of the JNK isoforms. This activation was blocked by expression of the MAP kinase phosphatase MKP-1. Comparison of the binding activity of the JNK isoforms demonstrated that the JNK proteins differ in their interaction with ATF2, Elk-1 and Jun transcription factors. Individual members of the JNK group may therefore selectively target specific transcription factors in vivo.
Role of the Raf/mitogen-activated protein kinase pathway in p21ras desensitizationKlarlund, Jes K.; Cherniack, Andrew D.; McMahon, Martin; Czech, Michael P. (1996-07-12)Desensitization of p21(ras) after stimulation of cells by growth factors and phorbol 12-myristate 13-acetate (PMA) correlates with hyperphosphorylation of the guanine nucleotide exchange factor Son-of-sevenless (Sos) and its dissociation from the adaptor protein Grb2 (Cherniack, A., Klarlund, J. K., Conway, B. R., and Czech, M. P. (1995) J. Biol. Chem. 270, 1485-1488). To test the role of the Raf/mitogen-activated protein (MAP) kinase pathway, we utilized cells expressing a chimera composed of the catalytic domain of p74Raf-1 and the hormone binding domain of the estradiol receptor (DeltaRaf-1:ER). Estradiol markedly stimulated DeltaRaf-1:ER and the downstream MEK and MAP kinases in these cells as well as Sos phosphorylation. However, the dissociation of Grb2 from Sos observed in response to PMA was not apparent upon DeltaRaf-1:ER activation. Furthermore, stimulation of DeltaRaf-1:ER did not impair GTP loading of p21(ras) in response to platelet-derived growth factor or epidermal growth factor. We conclude that activation of the Raf/MAP kinase pathway alone in these cells is insufficient to cause disassembly of Sos from Grb2 or to interrupt the ability of Sos to catalyze activation of p21(ras).