Constitutive activation of Mek1 by mutation of serine phosphorylation sites.

Huang, Weidong; Erikson, Raymond L.

doi:10.1073/pnas.91.19.8960

Cited by 140 publications

(121 citation statements)

References 30 publications

Supporting

Mentioning

116

Contrasting

Unclassified

Order By: Relevance

“…The replacement of regulatory phosphorylation sites by negatively charged residues from aspartate and glutamate to constitutively activate protein kinases has recently been used in the case of the MAPK kinase MEK1 (37)(38)(39)(40). Using this approach, it was possible to restore MEK activity independent of the upstream kinases and to analyze the role of activated MEK in growth, differentiation, and oncogenic transformation.…”

Section: Discussionmentioning

confidence: 99%

Constitutive Activation of Mitogen-activated Protein Kinase-activated Protein Kinase 2 by Mutation of Phosphorylation Sites and an A-helix Motif

Engel

Schultz

Martin

et al. 1995

Journal of Biological Chemistry

109

116

View full text Add to dashboard Cite

A recently described downstream target of mitogenactivated protein kinases (MAPKs) is the MAPK-activated protein (MAPKAP) kinase 2 which has been shown to be responsible for small heat shock protein phosphorylation. We have analyzed the mechanism of MAPKAP kinase 2 activation by MAPK phosphorylation using a recombinant MAPKAP kinase 2-fusion protein, p44 MAPK and p38/40 MAPK in vitro and using an epitope-tagged MAPKAP kinase 2 in heat-shocked NIH 3T3 cells. It is demonstrated that, in addition to the known phosphorylation of the threonine residue carboxyl-terminal to the catalytic domain, Thr-317, activation of MAPKAP kinase 2 in vitro and in vivo is dependent on phosphorylation of a second threonine residue, Thr-205, which is located within the catalytic domain and which is highly conserved in several protein kinases. Constitutive activation of MAPKAP kinase 2 is obtained by replacement of both of these threonine residues by glutamic acid. A constitutively active form of MAPKAP kinase 2 is also obtained by deletion of a carboxyl-terminal region containing Thr-317 and the A-helix motif or by replacing the conserved residues of the A-helix. These data suggest a dual mechanism of MAPKAP kinase 2 activation by phosphorylation of Thr-205 inside the catalytic domain and by phosphorylation of Thr-317 outside the catalytic domain involving an autoinhibitory A-helix motif.The network of mitogen-activated protein kinases is based on subsequent activation of protein kinases by phosphorylation (for a recent review, see Ref. 1). A major activator of the vertebrate MAP 1 kinases ERK1 and ERK2 has been identified as the protein kinase MEK, a dual specific kinase which itself is activated by protein kinases encoded by the proto-oncogenes raf1 or mos (2, 3) as well as by MEK kinase (4). In addition, stress-dependent signaling seems to proceed via parallel MAPK cascades leading to activation of further subgroups of MEKs and MAPKs (5, 6). One of the MAPK subgroups is designated stress-activated protein kinases (SAPKs) (7) and also termed amino-terminal c-Jun kinases (JNKs) (8,9). Another distinct subgroup covers the p38 MAPK and p40 MAPK , including the reactivating kinase (RK) (10 -12), which are more similar to the yeast MAPK homologue HOG1 (13).Signaling downstream of the MAPKs proceeds by phosphorylation of several transcription factors and of at least two different groups of MAPK-activated protein (MAPKAP) kinases, the different isoforms of ribosomal S6 kinase II (RSK, MAPKAP kinase 1) and the MAPKAP kinase 2. The latter enzyme has been shown to be activated by the MAPK ERK1 and ERK2 (14) in vitro and by the p38/40 MAPK (RK) in vivo (11, 12). Interestingly, activation of this kinase seems to be correlated to the phosphorylation of a threonine residue in a MAP kinase recognition consensus sequence PXTP located carboxylterminal to the catalytic domain of the enzyme (14). This would indicate a process of activation of MAPKAP kinase 2 different from other protein kinases, which are activated by phosphorylation within the catalyt...

show abstract

Section: Discussionmentioning

confidence: 99%

Constitutive Activation of Mitogen-activated Protein Kinase-activated Protein Kinase 2 by Mutation of Phosphorylation Sites and an A-helix Motif

Engel

Schultz

Martin

et al. 1995

Journal of Biological Chemistry

109

116

View full text Add to dashboard Cite

show abstract

“…MEKs are activated by phosphorylation of serine and threonine residues in their activation loop by upstream MEK kinases such as Raf [52,53]. Potent, constitutively-active versions of MEK1, MEK2, MKK3 and MKK6 can be created by the mutation of these residues to aspartate or glutamate, thereby mimicking the negative charge entailed by phosphorylation [34,36,54,55]. As shown in Fig.…”

Section: Deletion Of Docking Sites From Constitutively-active Mek Promentioning

confidence: 99%

Mitogen-activated protein kinase (MAPK)-docking sites in MAPK kinases function as tethers that are crucial for MAPK regulation in vivo

Grewal

Molina

Bardwell

2006

Cellular Signalling

View full text Add to dashboard Cite

Docking sites on targets of mitogen-activated protein kinases (MAPKs) facilitate accurate and efficient substrate phosphorylation. MAPK/ERK kinases (MEKs, or MKKs), the upstream regulators of MAPKs, also contain N-terminal MAPK-docking sites, or 'D-sites'; however, the in vivo functions of MEK D-sites are incompletely understood. Here we found that the ability of constitutively-active human MEK1 and MEK2 to stimulate ERK phosphorylation and to induce the neoplastic transformation of NIH 3T3 cells required the integrity of the D-site. In addition, Dsite mutants of otherwise wild-type MEK1/2 were unable to anchor unphosphorylated ERK2 in the cytoplasm. ERK activation, cytoplasmic anchoring and release were completely retained in 'swap' mutants in which MEK2's D-site was replaced with the D-site of MEK1 or yeast Ste7. Furthermore, these abilities were significantly retained when MEK2's D-site was moved to its Cterminus, or replaced by an unrelated MAPK-binding domain taken from the Ets-1 transcription factor. We conclude that the D-sites in MEKs are crucial for the activation of their cognate MAPKs in vivo, and that their primary function is to tether their cognate MAPKs near the MEK's kinase domain. This proximity effect is sufficient to explain the contribution that the D-site interaction makes to several biologically important signaling events.

show abstract

“…Fifty micrograms of recombinant GST-ERK-1 produced in bacteria was incubated with 10 g of recombinant M2-FLAG-MEK-1 S218/222D (a constitutively active form of MEK-1; Huang and Erikson, 1994) produced in COS-7 cells and precoupled to protein A/G beads, in a 100-l kinase assay buffer containing 100 M ATP for 60 min at 30°C. After the incubation, the beads were spun down, and the supernatant containing activated ERK-1 was collected.…”

Section: Erk-1 Activation In Vitromentioning

confidence: 99%

Identification of Novel In Vivo Raf-1 Phosphorylation Sites Mediating Positive Feedback Raf-1 Regulation by Extracellular Signal-regulated Kinase

Balan

Leicht

Zhu³

et al. 2006

MBoC

View full text Add to dashboard Cite

The Ras-Raf-mitogen-activated protein kinase cascade is a key growth-signaling pathway, which uncontrolled activation results in transformation. Although the exact mechanisms underlying Raf-1 regulation remain incompletely understood, phosphorylation has been proposed to play a critical role in this regulation. We report here three novel epidermal growth factor-induced in vivo Raf-1 phosphorylation sites that mediate positive feedback Raf-1 regulation. Using mass spectrometry, we identified Raf-1 phosphorylation on three SP motif sites: S289/S296/S301 and confirmed their identity using twodimensional-phosphopeptide mapping and phosphospecific antibodies. These sites were phosphorylated by extracellular signal-regulated kinase (ERK)-1 in vitro, and their phosphorylation in vivo was dependent on endogenous ERK activity. Functionally, ERK-1 expression sustains Raf-1 activation in a manner dependent on Raf-1 phosphorylation on the identified sites, and S289/296/301A substitution markedly decreases the in vivo activity of Raf-1 S259A. Importantly, the ERKphosphorylated Raf-1 pool has 4 times higher specific kinase activity than total Raf-1, and its phosphopeptide composition is similar to that of the general Raf-1 population, suggesting that the preexisting, phosphorylated Raf-1, representing the activatable Raf-1 pool, is the Raf-1 subpopulation targeted by ERK. Our study describes the identification of new in vivo Raf-1 phosphorylation sites targeted by ERK and provides a novel mechanism for a positive feedback Raf-1 regulation. INTRODUCTIONRaf-1 is part of the Ras-Raf-mitogen-activated protein kinase kinase (MEK)-mitogen-activated protein kinase (MAPK) pathway, among the first mammalian signaling cascades to be elucidated (Avruch et al., 2001;Chang and Karin, 2001). The Ras-Raf-MAPK pathway is responsible for transmitting signals from membrane-bound receptors to intracellular and to nuclear targets, coordinating cellular response to a variety of environmental factors (Liebmann, 2001;Pearson et al., 2001). Aberrations at the receptor level and along the Ras-Raf-MAPK pathway are associated with a variety of diseases, especially cancer, with mutations in Ras detected in Ͼ30% of all human cancers and reaching frequencies of up to 50 -90% in specific carcinomas (Porter and Vaillancourt, 1998;Lyons et al., 2001;Herrera and Sebolt-Leopold, 2002). Recently, also activating mutations in the B-Raf gene have been identified in various cancers, most predominantly melanomas (Davies et al., 2002;Mercer and Pritchard, 2003). In addition, both Ras and Raf are protooncogenes that occur naturally as viral-transmitted oncoproteins (Rapp et al., 1983). The initial elucidation of the Ras-Raf-MAPK pathway resulted from complimentary molecular and biochemical studies in mammalian cells and genetic studies in yeast, Drosophila and Caenorhabditis elegans (Avruch et al., 2001). Although the general features of the pathway and its activation mode are known, the exact regulatory mechanisms, at the molecular level, remain incompletely underst...

show abstract

Constitutive activation of Mek1 by mutation of serine phosphorylation sites.

Cited by 140 publications

References 30 publications

Constitutive Activation of Mitogen-activated Protein Kinase-activated Protein Kinase 2 by Mutation of Phosphorylation Sites and an A-helix Motif

Constitutive Activation of Mitogen-activated Protein Kinase-activated Protein Kinase 2 by Mutation of Phosphorylation Sites and an A-helix Motif

Mitogen-activated protein kinase (MAPK)-docking sites in MAPK kinases function as tethers that are crucial for MAPK regulation in vivo

Identification of Novel In Vivo Raf-1 Phosphorylation Sites Mediating Positive Feedback Raf-1 Regulation by Extracellular Signal-regulated Kinase

Contact Info

Product

Resources

About