scholarly journals Identification of Raf-1 S471 as a Novel Phosphorylation Site Critical for Raf-1 and B-Raf Kinase Activities and for MEK Binding

2005 ◽  
Vol 16 (10) ◽  
pp. 4733-4744 ◽  
Author(s):  
Jun Zhu ◽  
Vitaly Balan ◽  
Agnieszka Bronisz ◽  
Karina Balan ◽  
Hengrui Sun ◽  
...  
Keyword(s):  
Kinase Activity ◽  
Cell Transformation ◽  
Signal Transmission ◽  
Phosphorylation Site ◽  
Kinase Domain ◽  
Mitogen Activated Protein Kinase ◽  
Phosphorylation Sites ◽  
Raf Kinase ◽  
Mitogen Activated Protein

The Ras-Raf-MAPK cascade is a key growth-signaling pathway and its uncontrolled activation results in cell transformation. Although the general features of the signal transmission along the cascade are reasonably defined, the mechanisms underlying Raf activation remain incompletely understood. Here, we show that Raf-1 dephosphorylation, primarily at epidermal growth factor (EGF)-induced sites, abolishes Raf-1 kinase activity. Using mass spectrometry, we identified five novel in vivo Raf-1 phosphorylation sites, one of which, S471, is located in subdomain VIB of Raf-1 kinase domain. Mutational analyses demonstrated that Raf-1 S471 is critical for Raf-1 kinase activity and for its interaction with mitogen-activated protein kinase kinase (MEK). Similarly, mutation of the corresponding B-Raf site, S578, resulted in an inactive kinase, suggesting that the same Raf-1 and B-Raf phosphorylation is needed for Raf kinase activation. Importantly, the naturally occurring, cancer-associated B-Raf activating mutation V599E suppressed the S578A mutation, suggesting that introducing a charged residue at this region eliminates the need for an activating phosphorylation. Our results demonstrate an essential role of specific EGF-induced Raf-1 phosphorylation sites in Raf-1 activation, identify Raf-1 S471 as a novel phosphorylation site critical for Raf-1 and B-Raf kinase activities, and point to the possibility that the V599E mutation activates B-Raf by mimicking a phosphorylation at the S578 site.

scholarly journals Role for the Ran Binding Protein, Mog1p, in Saccharomyces cerevisiae SLN1-SKN7 Signal Transduction

2004 ◽  
Vol 3 (6) ◽  
pp. 1544-1556 ◽  
Author(s):  
Jade Mei-Yeh Lu ◽  
Robert J. Deschenes ◽  
Jan S. Fassler
Keyword(s):  
Signal Transduction ◽  
Transcription Factor ◽  
Osmotic Stress ◽  
Kinase Activity ◽  
Mitogen Activated Protein Kinase ◽  
Osmotic Response ◽  
Mitogen Activated Protein ◽  
Kinase Pathway

ABSTRACT Yeast Sln1p is an osmotic stress sensor with histidine kinase activity. Modulation of Sln1 kinase activity in response to changes in the osmotic environment regulates the activity of the osmotic response mitogen-activated protein kinase pathway and the activity of the Skn7p transcription factor, both important for adaptation to changing osmotic stress conditions. Many aspects of Sln1 function, such as how kinase activity is regulated to allow a rapid response to the continually changing osmotic environment, are not understood. To gain insight into Sln1p function, we conducted a two-hybrid screen to identify interactors. Mog1p, a protein that interacts with the yeast Ran1 homolog, Gsp1p, was identified in this screen. The interaction with Mog1p was characterized in vitro, and its importance was assessed in vivo. mog1 mutants exhibit defects in SLN1-SKN7 signal transduction and mislocalization of the Skn7p transcription factor. The requirement for Mog1p in normal localization of Skn7p to the nucleus does not fully account for the mog1-related defects in SLN1-SKN7 signal transduction, raising the possibility that Mog1p may play a role in Skn7 binding and activation of osmotic response genes.

scholarly journals EGF stimulates gastrin promoter through activation of Sp1 kinase activity

2000 ◽  
Vol 278 (4) ◽  
pp. C697-C708 ◽  
Author(s):  
Sergey Chupreta ◽  
Ming Du ◽  
Andrea Todisco ◽  
Juanita L. Merchant
Keyword(s):  
Kinase Activity ◽  
Egf Receptor ◽  
Solid Phase ◽  
Receptor Activation ◽  
Mitogen Activated Protein Kinase ◽  
Kinase Assay ◽  
Ags Cells ◽  
Extracellular Signal ◽  
Mitogen Activated Protein

Epidermal growth factor (EGF) receptor activation stimulates gastrin gene expression through a GC-rich element called gastrin EGF response element (gERE). This element is bound by Sp1 family members and is a target of the ras-extracellular signal-regulated kinase (Erk) signal transduction cascade. This raised the possibility that Sp1 may be phosphorylated by kinases of this signaling pathway. Erk is capable of phosphorylating other mitogen-inducible transcription factors, e.g., Elk and Sap, suggesting that Erk may also mediate EGF-dependent phosphorylation of Sp1. This possibility was tested by studying Sp1-dependent kinase activity in extracts prepared from EGF-activated AGS cells by use of solid-phase kinase assays and immunoprecipitation of metabolically labeled Sp1. The results revealed that Sp1 kinase activity (like gastrin promoter activation) is inhibited by PD-98059 and, therefore, is dependent on mitogen-activated protein kinase kinase 1 (Mek 1). However, EGF-dependent activation of endogenous Erk did not account for most of the Sp1 kinase activity, since Erk and additional Sp1 kinase activity analyzed in a solid-phase kinase assay eluted from an ion-exchange column in different fractions. Phosphoamino acid analysis of in vivo radiolabeled Sp1 demonstrated that the kinase phosphorylates Sp1 on Ser and Thr in response to EGF. Therefore, most EGF-stimulated Sp1 kinase activity is Mek 1 dependent and distinct from Erk.

scholarly journals FAK signaling is critical for ErbB-2/ErbB-3 receptor cooperation for oncogenic transformation and invasion

2005 ◽  
Vol 171 (3) ◽  
pp. 505-516 ◽  
Author(s):  
Naciba Benlimame ◽  
Qiang He ◽  
Su Jie ◽  
Dingzhang Xiao ◽  
Ying Jie Xu ◽  
...  
Keyword(s):  
Cancer Progression ◽  
Cell Invasion ◽  
Cell Transformation ◽  
Mitogen Activated Protein Kinase ◽  
Tyrosine Kinase Receptor ◽  
Oncogenic Transformation ◽  
Mitogen Activated Protein ◽  
Migratory Cells

The overexpression of members of the ErbB tyrosine kinase receptor family has been associated with cancer progression. We demonstrate that focal adhesion kinase (FAK) is essential for oncogenic transformation and cell invasion that is induced by ErbB-2 and -3 receptor signaling. ErbB-2/3 overexpression in FAK-deficient cells fails to promote cell transformation and rescue chemotaxis deficiency. Restoration of FAK rescues both oncogenic transformation and invasion that is induced by ErbB-2/3 in vitro and in vivo. In contrast, the inhibition of FAK in FAK-proficient invasive cancer cells prevented cell invasion and metastasis formation. The activation of ErbB-2/3 regulates FAK phosphorylation at Tyr-397, -861, and -925. ErbB-induced oncogenic transformation correlates with the ability of FAK to restore ErbB-2/3–induced mitogen-activated protein kinase (MAPK) activation; the inhibition of MAPK prevented oncogenic transformation. In contrast, the inhibition of Src but not MAPK prevented ErbB–FAK-induced chemotaxis. In migratory cells, activated ErbB-2/3 receptors colocalize with activated FAK at cell protrusions. This colocalization requires intact FAK. In summary, distinct FAK signaling has an essential function in ErbB-induced oncogenesis and invasiveness.

Phosphorylation of Argonaute 2 at serine-387 facilitates its localization to processing bodies

2008 ◽  
Vol 413 (3) ◽  
pp. 429-436 ◽  
Author(s):  
Yan Zeng ◽  
Heidi Sankala ◽  
Xiaoxiao Zhang ◽  
Paul R. Graves
Keyword(s):  
Protein Kinase ◽  
P38 Mapk ◽  
Kinase Inhibitor ◽  
Mapk Pathway ◽  
Phosphorylation Site ◽  
Mitogen Activated Protein Kinase ◽  
Processing Bodies ◽  
Mitogen Activated Protein

Ago (Argonaute) proteins are essential effectors of RNA-mediated gene silencing. To explore potential regulatory mechanisms for Ago proteins, we examined the phosphorylation of human Ago2. We identified serine-387 as the major Ago2 phosphorylation site in vivo. Phosphorylation of Ago2 at serine-387 was significantly induced by treatment with sodium arsenite or anisomycin, and arsenite-induced phosphorylation was inhibited by a p38 MAPK (mitogen-activated protein kinase) inhibitor, but not by inhibitors of JNK (c-Jun N-terminal kinase) or MEK [MAPK/ERK (extracellular-signal-regulated kinase) kinase]. MAPKAPK2 (MAPK-activated protein kinase-2) phosphorylated bacterially expressed full-length human Ago2 at serine-387 in vitro, but not the S387A mutant. Finally, mutation of serine-387 to an alanine residue or treatment of cells with a p38 MAPK inhibitor reduced the localization of Ago2 to processing bodies. These results suggest a potential regulatory mechanism for RNA silencing acting through Ago2 serine-387 phosphorylation mediated by the p38 MAPK pathway.

scholarly journals Met/Hepatocyte Growth Factor Receptor Ubiquitination Suppresses Transformation and Is Required for Hrs Phosphorylation

2005 ◽  
Vol 25 (21) ◽  
pp. 9632-9645 ◽  
Author(s):  
Jasmine V. Abella ◽  
Pascal Peschard ◽  
Monica A. Naujokas ◽  
Tong Lin ◽  
Caroline Saucier ◽  
...  
Keyword(s):  
Cell Transformation ◽  
Growth Factor Receptor ◽  
Mitogen Activated Protein Kinase ◽  
Endosomal Trafficking ◽  
Met Receptor ◽  
Down Regulation ◽  
Lysosomal Sorting ◽  
Mitogen Activated Protein ◽  
Sustained Activation

ABSTRACT The Met receptor tyrosine kinase (RTK) regulates epithelial remodeling, dispersal, and invasion and is deregulated in many human cancers. It is now accepted that impaired down-regulation, as well as sustained activation, of RTKs could contribute to their deregulation. Down-regulation of the Met receptor involves ligand-induced internalization, ubiquitination by Cbl ubiquitin ligases, and lysosomal degradation. Here we report that a ubiquitination-deficient Met receptor mutant (Y1003F) is tumorigenic in vivo. The Met Y1003F mutant is internalized, and undergoes endosomal trafficking with kinetics similar to the wild-type Met receptor, yet is inefficiently targeted for degradation. This results in sustained activation of Met Y1003F and downstream signals involving the Ras-mitogen-activated protein kinase pathway, cell transformation, and tumorigenesis. Although Met Y1003F undergoes endosomal trafficking and localizes with the cargo-sorting protein Hrs, it is unable to induce phosphorylation of Hrs. Fusion of monoubiquitin to Met Y1003F is sufficient to decrease Met receptor stability and prevent sustained MEK1/2 activation. In addition, this rescues Hrs tyrosine phosphorylation and decreases transformation in a focus-forming assay. These results demonstrate that Cbl-dependent ubiquitination is dispensable for Met internalization but is critical to target the Met receptor to components of the lysosomal sorting machinery and to suppress its inherent transforming activity.

scholarly journals Negative regulation of Raf-1 by phosphorylation of serine 621.

1996 ◽  
Vol 16 (10) ◽  
pp. 5409-5418 ◽  
Author(s):  
H Mischak ◽  
T Seitz ◽  
P Janosch ◽  
M Eulitz ◽  
H Steen ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Kinase Activity ◽  
Phosphorylation Site ◽  
Kinase Domain ◽  
Negative Regulation ◽  
Catalytic Function ◽  
Dependent Protein ◽  
The One

The elevation of cyclic AMP (cAMP) levels in the cell downregulates the activity of the Raf-1 kinase. It has been suggested that this effect is due to the activation of cAMP-dependent protein kinase (PKA), which can directly phosphorylate Raf-1 in vitro. In this study, we confirmed this hypothesis by coexpressing Raf-1 with the constitutively active catalytic subunit of PKA, which could fully reproduce the inhibition previously achieved by cAMP. PKA-phosphorylated Raf-1 exhibits a reduced affinity for GTP-loaded Ras as well as impaired catalytic activity. As the binding to GTP-loaded Ras induces Raf-1 activation in the cell, we examined which mechanism is required for PKA-mediated Raf-1 inhibition in vivo. A Raf-1 point mutant (RafR89L), which is unable to bind Ras, as well as the isolated Raf-1 kinase domain were still fully susceptible to inhibition by PKA, demonstrating that the phosphorylation of the Raf-1 kinase suffices for inhibition. By the use of mass spectroscopy and point mutants, PKA phosphorylation site was mapped to a single site in the Raf-1 kinase domain, serine 621. Replacement of serine 621 by alanine or cysteine or destruction of the PKA consensus motif by changing arginine 618 resulted in the loss of catalytic activity. Notably, a mutation of serine 619 to alanine did not significantly affect kinase activity or regulation by activators or PKA. Changing serine 621 to aspartic acid yielded a Raf-1 protein which, when expressed to high levels in Sf-9 insect cells, retained a very low inducible kinase activity that was resistant to PKA downregulation. The purified Raf-1 kinase domain displayed slow autophosphorylation of serine 621, which correlated with a decrease in catalytic function. The Raf-1 kinase domain activated by tyrosine phosphorylation could be downregulated by PKA. Specific removal of the phosphate residue at serine 621 reactivated the catalytic activity. These results are most consistent with a dual role of serine 621. On the one hand, serine 621 appears essential for catalytic activity; on the other hand, it serves as a phosphorylation site which confers negative regulation.

scholarly journals Mitogen-activated protein kinase kinase 1 (MKK1) is negatively regulated by threonine phosphorylation

1994 ◽  
Vol 14 (3) ◽  
pp. 1594-1602
Author(s):  
A J Rossomando ◽  
P Dent ◽  
T W Sturgill ◽  
D R Marshak
Keyword(s):  
Protein Kinase ◽  
Phosphorylation Site ◽  
Mitogen Activated Protein Kinase ◽  
Dual Specificity ◽  
Mitogen Activated Protein ◽  
Kinase Cascade ◽  
Protein Kinase Cascade ◽  
Protein Kinase Kinase

Mitogen-activated protein kinase kinase 1 (MKK1), a dual-specificity tyrosine/threonine protein kinase, has been shown to be phosphorylated and activated by the raf oncogene product as part of the mitogen-activated protein kinase cascade. Here we report the phosphorylation and inactivation of MKK1 by phosphorylation on threonine 286 and threonine 292. MKK1 contains a consensus phosphorylation site for p34cdc2, a serine/threonine protein kinase that regulates the cell division cycle, at Thr-286 and a related site at Thr-292. p34cdc2 catalyzes the in vitro phosphorylation of MKK1 on both of these threonine residues and inactivates MKK1 enzymatic activity. Both sites are phosphorylated in vivo as well. The data presented in this report provide evidence that MKK1 is negatively regulated by threonine phosphorylation.

scholarly journals Mutational Activation of ErbB2 Reveals a New Protein Kinase Autoinhibition Mechanism

2007 ◽  
Vol 283 (3) ◽  
pp. 1588-1596 ◽  
Author(s):  
Ying-Xin Fan ◽  
Lily Wong ◽  
Jinhui Ding ◽  
Nikolay A. Spiridonov ◽  
Richard C. Johnson ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Human Cancer ◽  
Kinase Domain ◽  
Mitogen Activated Protein Kinase ◽  
Protein Kinase Activity ◽  
Mitogen Activated Protein ◽  
Cancer Mutations ◽  
Mutational Activation ◽  
New Protein

Autoinhibition plays a key role in the control of protein kinase activity. ErbB2 is a unique receptor-tyrosine kinase that does not bind ligand but possesses an extracellular domain poised to engage other ErbBs. Little is known about the molecular mechanism for ErbB2 catalytic regulation. Here we show that ErbB2 kinase is strongly autoinhibited, and a loop connecting the αC helix and β4 sheet within the kinase domain plays a major role in the control of kinase activity. Mutations of two Gly residues at positions 776 and 778 in this loop dramatically increase ErbB2 catalytic activity. Kinetic analysis demonstrates that mutational activation is due to ∼10- and ∼7-fold increases in ATP binding affinity and turnover number, respectively. Expression of the activated ErbB2 mutants in cells resulted in elevated ligand-independent ErbB2 autophosphorylation, ErbB3 phosphorylation, and stimulation of mitogen-activated protein kinase. Molecular modeling suggests that the ErbB2 kinase domain is stabilized in an inactive state via a hydrophobic interaction between the αC-β4 and activation loops. Importantly, many ErbB2 human cancer mutations have been identified in the αC-β4 loop, including the activating G776S mutation studied here. Our findings reveal a new kinase regulatory mechanism in which the αC-β4 loop functions as an intramolecular switch that controls ErbB2 activity and suggests that loss of αC-β4 loop-mediated autoinhibition is involved in oncogenic activation of ErbB2.

scholarly journals Characterization and partial purification of a novel neutrophil membrane-associated kinase capable of phosphorylating the respiratory burst component p47phox

1999 ◽  
Vol 338 (2) ◽  
pp. 359-366 ◽  
Author(s):  
Aroon S. LAL ◽  
Peter J. PARKER ◽  
Anthony W. SEGAL
Keyword(s):  
Protein Kinase ◽  
Respiratory Burst ◽  
Kinase Activity ◽  
Gel Filtration ◽  
Mitogen Activated Protein Kinase ◽  
Partial Purification ◽  
Exact Nature ◽  
Mitogen Activated Protein ◽  
Respiratory Burst Oxidase

The phosphorylation of p47phox is widely viewed as an important step in the activation of the neutrophil respiratory burst oxidase system. The exact nature of the kinase(s) responsible remains to be elucidated. We show here that such a kinase was detected on neutrophil membranes activated by either PMA or formyl-methionyl-leucyl-phenylalanine. This enzyme is not intrinsic to the neutrophil membrane and could be eluted with 0.5 M NaCl. The kinase activity was partially purified and was found not to be due to the presence of previously suggested kinases, including protein kinase C isotypes, mitogen-activated protein kinase and protein kinase B. Gel filtration and renaturation in substrate gels suggest a molecular mass of between 45 and 51 kDa. The kinase activity was independent of calcium and lipids but was potently inhibited by staurosporine. Treatment with protein phosphatase 2Ac suggested that the kinase was activated by serine/threonine phosphorylation. Phosphopeptide maps indicated that the kinase phosphorylated p47phox on similar sites to those found in vivo. These results indicate that activation of neutrophils by PMA results in the activation of a membrane-associated kinase that may play a part in the regulation of neutrophil NADPH oxidase through its ability to phosphorylate p47phox.

scholarly journals Identification of the mitogen-activated protein kinase phosphorylation sites on human Sos1 that regulate interaction with Grb2.

1996 ◽  
Vol 16 (10) ◽  
pp. 5674-5682 ◽  
Author(s):  
S Corbalan-Garcia ◽  
S S Yang ◽  
K R Degenhardt ◽  
D Bar-Sagi
Keyword(s):  
Growth Factor ◽  
Map Kinase ◽  
Tyrosine Kinases ◽  
Phosphorylation Site ◽  
Mitogen Activated Protein Kinase ◽  
Phosphorylation Sites ◽  
Nucleotide Exchange ◽  
Mitogen Activated Protein ◽  
Son Of Sevenless ◽  
Kinase Phosphorylation

The Son of sevenless proteins (Sos) are guanine nucleotide exchange factors involved in the activation of Ras by cytoplasmic and receptor tyrosine kinases. Growth factor stimulation rapidly induces the phosphorylation of Sos on multiple serine and threonine sites. Previous studies have demonstrated that growth factor-induced Sos phosphorylation occurs at the C-terminal region of the protein and is mediated, in part, by mitogen-activated protein (MAP) kinase. In this report, we describe the identification of five MAP kinase sites (S-1137, S-1167, S-1178, S-1193, and S-1197) on hSos1. We demonstrate that four of these sites, S-1132, S-1167, S-1178, and S-1193, become phosphorylated following growth factor stimulation. The MAP kinase phosphorylation sites are clustered within a region encompassing three proline-rich SH3-binding sites in the C-terminal domain of hSos1. Replacing the MAP kinase phosphorylation sites with alanine residues results in an increase in the binding affinity of Grb2 to hSos1. Interestingly, hSos2 contains only one MAP kinase phosphorylation site and, as demonstrated previously, has an increased affinity toward Grb2 compared with hSos1. These results suggest a role for MAP kinase in the regulation of Grb2-Sos interactions. Since the binding of Grb2 is important for Sos function, the phosphorylation-dependent modulation of Grb2-Sos association may provide a means of controlling Ras activation.

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