Docking Motif Interactions in MAP Kinases Revealed by Hydrogen Exchange Mass Spectrometry

Lee, Thomas; Hoofnagle, Andrew N.; Kabuyama, Yukihito; Stroud, James C.; Min, Xiaoshan; Goldsmith, Elizabeth J.; Chen, Lin; Resing, Katheryn A.; Ahn, Natalie G.

doi:10.1016/s1097-2765(04)00161-3

Cited by 279 publications

(366 citation statements)

References 33 publications

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“…Previously, QKGRKPRDLELPLSPSL, which is derived from Elk-1, had been shown by HX-MS studies to bind the DRS of ERK2 (36). Figure 6 shows several SDS-PAGE gels used to fractionate NTCB-mediated cleavage products of ERK2-CTs after alkylation in the absence and presence of a saturating concentration (625 μM) of the D-site peptide.…”

Section: Footprinting Reveals Binding Of Ets-1 and A D-site Peptide Imentioning

confidence: 99%

“…While the resolution of the HX-MS approach is limited to peptide fragments, it has the advantage of allowing a large fraction of a protein to be scanned relatively easily. In the case of ERK2, for example, it was possible to analyze almost 90% of the molecule (36). Thus, HX-MS provides the opportunity for wide coverage, but at a relatively low resolution.…”

Section: Comparison Of Cysteine Footprinting With Mutagenesis and Hx-msmentioning

confidence: 99%

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Expanding the Repertoire of an ERK2 Recruitment Site: Cysteine Footprinting Identifies the D-Recruitment Site as a Mediator of Ets-1 Binding

et al. 2007

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Many substrates of ERK2 contain a D-site, a sequence recognized by ERK2 that is used to promote catalysis. Despite lacking a canonical D-site, the substrate Ets-1 is displaced from ERK2 by peptides containing one. This suggests that Ets-1 may contain a novel or cryptic D-site. To investigate this possibility a protein footprinting strategy was developed to elucidate ERK2-ligand interactions. Using this approach, single cysteine reporters were placed in the D-recruitment site (DRS) of ERK2 and the resulting ERK2 proteins subjected to alkylation by iodoacetamide. The ability of residues 1-138 of Ets-1 to protect the cysteines from alkylation was determined. The pattern of protection observed is consistent with Ets-1 occupying a hydrophobic binding site within the DRS of ERK2. Significantly, a peptide derived from the D-site of Elk-1, which is known to bind the DRS, exhibits a similar pattern of cysteine protection. This analysis expands the repertoire of the DRS on ERK2 and suggests that other targeting sequences remain to be identified. Furthermore, cysteinefootprinting is presented as a useful way to interrogate protein-ligand interactions at the resolution of a single amino acid.The mitogen-activated protein kinases (MAPKs) 1 are ubiquitous elements of eukaryotic signaling pathways that permeate nearly all aspects of signaling in multicellular organisms. In humans the loss of their regulation is associated with many diseases that encompass an expanding list of cancers as well as neurological and inflammatory diseases (1). Three main subgroups have been identified in humans, termed the ERKs (2), the JNKs (3,4), and the p38 MAPKs (5,6). Several newer members have recently been reviewed (7). Extracellular regulated protein kinase 2 (ERK2), the prototypical member of the MAP kinase family, catalyzes the † This research was supported in part by the Welch Foundation (F-1390) and the National Institutes of Health (GM59802). Mass spectra were acquired by Dr. Herng-Hsiang Lo in the CRED Analytical Instrumentation Facility Core supported by the NIEHS center grant ES07784. Molecular graphics images were produced using the UCSF Chimera package from the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (supported by NIH P41 RR-01081). NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 July 6. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript transfer of the γ-phosphate of adenosine triphosphate to serine or threonine residues that generally lie in flexible regions of proteins. It is a remarkable enzyme whose ability to exhibit high specificity toward a discrete subset of structurally diverse proteins is poorly understood.Protein kinases generally recognize substrates through a consensus sequence that contains the phosphorylation site (8). This sequence is recognized, in part, by a region called the activation segment that encompasses residues 165 DFG to APE 195 of the catalytic domain of protein ki...

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Section: Footprinting Reveals Binding Of Ets-1 and A D-site Peptide Imentioning

confidence: 99%

Section: Comparison Of Cysteine Footprinting With Mutagenesis and Hx-msmentioning

confidence: 99%

Expanding the Repertoire of an ERK2 Recruitment Site: Cysteine Footprinting Identifies the D-Recruitment Site as a Mediator of Ets-1 Binding

et al. 2007

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“…Note Added in Proof-The binding surface of ERK for the DEF domain was determined by Lee et al (48). The surface was reported to be exposed after ERK is activated.…”

mentioning

confidence: 99%

Extracellular Signal-regulated Kinase Activated by Epidermal Growth Factor and Cell Adhesion Interacts with and Phosphorylates Vinexin

Mitsushima

Suwa

Amachi

et al. 2004

Journal of Biological Chemistry

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Extracellular signal-regulated kinase 1/2 (ERK1/2) is activated by various extracellular stimuli including growth factors and cytokines and plays a pivotal role in regulating cell proliferation and differentiation by phosphorylating nuclear transcription factors. Recently, it was reported that activated ERK1/2 also concentrates at adhesion sites and regulates cell spreading and migration. Vinexin is a focal adhesion protein regulating both cell spreading and growth factor signaling. We show here that vinexin was directly phosphorylated by ERK1/2 upon stimulation with growth factors. ERK1/2 phosphorylated the linker region of vinexin between the second and third SH3 domains. Site-directed mutagenesis revealed that ERK2 mainly phosphorylated the serine 189 residue of vinexin ␤. Furthermore, vinexin ␤ interacted with ERK1/2 both in vitro and in vivo. Vinexin interacted with the active but not inactive form of ERK1/2. A putative DEF (docking for ERK FXFP) domain located in the linker region of vinexin was required for the interaction with ERK1/2 and efficient phosphorylation of vinexin ␤ by ERK2. Finally, we showed that cell adhesion to fibronectin also induced the association of vinexin ␤ with ERK2 and the phosphorylation of vinexin ␤. Furthermore, vinexin and ERK were co-localized to the periphery of cells during cell spreading on fibronectin. Together, these results suggest that vinexin is a novel substrate of ERK2 and may play roles in ERK-dependent cell regulation during cell spreading as well as in growth factor-induced responses.Mitogen-activated protein kinase (MAPK) 1 consists of four subfamilies, extracellular signal-regulated kinase 1/2 (ERK1/ 2), c-Jun N-terminal kinase/stress-activated kinase, p38MAPK, and ERK5. ERK1/2 is mainly activated by various growth factors and regulates a diverse array of cellular events including cell proliferation, survival, and differentiation (1, 2). Many extracellular signals activate membrane receptors, including receptor tyrosine kinases, G protein-coupled receptors, or integrins, leading to the activation of Raf. Activated Raf, in turn, activates MEK (MAPK/ERK kinase) by the direct phosphorylation of dual serine residues. Activated MEK also directly phosphorylates and activates ERK1/2. Once activated, ERK1/2 enters the nucleus and phosphorylates nuclear transcription factors, including Elk-1, Sap1, c-Myc, and c-Fos (3).Recently, ERK has been shown to play crucial roles in regulating cell adhesion and cell migration independent of its nuclear functions (4 -7). ERK is involved in the migration of breast cancer cells stimulated by urokinase-type tissue plasminogen activator. Neither de novo gene transcription nor protein synthesis is required for this process (5). Activation of ERK is also suggested to be involved in the chemotaxis or the extension of pseudopodia, probably through the phosphorylation of cytoplasmic proteins (8, 9). Furthermore, ERK is well known to be activated by integrin engagement and to be localized to adhesion sites (7, 10 -14). Although some cytoplasm...

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“…42,43 Such docking sites have been shown to be important for substrate recognition by a number of protein kinases, including several MAP kinases. [44][45][46] In addition, different protein kinases have been found to recognize similar, or overlapping, consensus sequences. 47 Therefore, it would be useful to have an additional filter that could help identify those consensus elements that might represent bona fide sites of phosphorylation.…”

Section: Do Not Distributementioning

confidence: 99%

Identifying Atg1 Substrates: Four Means to an End

Deminoff

Herman²

2007

Autophagy

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Docking Motif Interactions in MAP Kinases Revealed by Hydrogen Exchange Mass Spectrometry

Cited by 279 publications

References 33 publications

Expanding the Repertoire of an ERK2 Recruitment Site: Cysteine Footprinting Identifies the D-Recruitment Site as a Mediator of Ets-1 Binding

Expanding the Repertoire of an ERK2 Recruitment Site: Cysteine Footprinting Identifies the D-Recruitment Site as a Mediator of Ets-1 Binding

Extracellular Signal-regulated Kinase Activated by Epidermal Growth Factor and Cell Adhesion Interacts with and Phosphorylates Vinexin

Identifying Atg1 Substrates: Four Means to an End

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