The Cytoskeletal/Non-muscle Isoform of α-Actinin Is Phosphorylated on Its Actin-binding Domain by the Focal Adhesion Kinase

Izaguirre, Gonzalo; Aguirre, Lina; Hu, Ya Ping; Lee, Hwa Young; Schlaepfer, David D.; Aneskievich, Brian J.; Haimovich, Beatrice

doi:10.1074/jbc.m101678200

Cited by 136 publications

(162 citation statements)

References 65 publications

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“…For example, ␣-actinin was detected in IMAC eluates and Tyr(P) IPs only after pV stimulation in agreement with published work (38,39) that indicates that this protein is reversibly phosphorylated. Tyr-12 phosphorylation of ␣-actinin, previously detected upon pV treatment (39), was not identified in our analyses.…”

Section: Identification Of Proteins Phosphorylated At Tyr Residues (Asupporting

confidence: 91%

Quantification of Gel-separated Proteins and Their Phosphorylation Sites by LC-MS Using Unlabeled Internal Standards

Cutillas

Geering

Waterfield

et al. 2005

Molecular & Cellular Proteomics

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Protein phosphorylation plays a critical role in normal cellular function and is often subverted in disease. Although major advances have recently been made in identification and quantitation of protein phosphorylation sites by MS, current methodological limitations still preclude routine, easily usable, and comprehensive quantitative analysis of protein phosphorylation. Here we report a simple LC-MS method to quantify gel-separated proteins and their sites of phosphorylation; in this approach, integrated chromatographic peak areas of peptide analytes from proteins under study are normalized to those of a nonisotopically labeled internal standard protein spiked into the excised gel samples just prior to in-gel digestion. The internal standard intensities correct for differences in enzymatic activities and sample losses that may occur during the processes of in-gel digestion and peptide extraction from the gel pieces. We used this method of peak area measurement with an internal standard to investigate the effects of pervanadate on protein phosphorylation in the WEHI-231 B cell lymphoma cell line and to assess the role of phosphoinositide 3-kinase (PI3K) in these phosphorylation events. Phosphoproteins, isolated from total cell lysates using IMAC or by immunoprecipitation using Tyr(P) antibodies, were analyzed using this method, leading to identification of >400 proteins, several of which were found at higher levels in phosphoprotein fractions after pervanadate treatment. Pretreatment of cells with the PI3K inhibitor wortmannin reduced the phosphorylation level of certain proteins (e.g. STAT1 and phospholipase C␥2) while increasing the phosphorylation of several others. Peak area measurement with an internal standard was also used to follow the dynamics of PI3K-dependent and -independent changes in the post-translational modification of both known and novel phospho- Reversible covalent modification of proteins is a key molecular mechanism by which membrane receptor activation is converted into intracellular signaling cascades that mediate physiological change. Engagement of ligands with their receptors results in rapid and transient phosphorylation of downstream protein targets, which as a consequence modify their catalytic properties, cellular localization, and/or binding partners. As a consequence, intracellular signaling pathways can control critical aspects of metabolism, cell cycle progression, migration, differentiation, and protein synthesis. Many diseases are a direct consequence of deregulation in these processes (1, 2); these changes may be detectable by quantitative phosphoprotein analysis.The assessment of the phosphorylation status of specific proteins is commonly performed by immunochemical methods using antisera raised against a peptide containing the site of phosphorylation of the target protein. This approach is restricted to the study of known phosphorylation sites and cannot be used to monitor and discover novel phosphorylated proteins and their sites of phosphorylation. To overcome this proble...

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Section: Identification Of Proteins Phosphorylated At Tyr Residues (Asupporting

confidence: 91%

Quantification of Gel-separated Proteins and Their Phosphorylation Sites by LC-MS Using Unlabeled Internal Standards

Cutillas

Geering

Waterfield

et al. 2005

Molecular & Cellular Proteomics

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“…Because of its localization in focal adhesions, ␣-actinin was suggested to anchor the actin filaments to the plasma membrane (52). Recently, FAK was found to phosphorylate ␣-actinin, thereby reducing the interactions between ␣-actinin and actin filaments (53). A decrease in the affinity of ␣-actinin for actin resulting from phosphorylation by FAK could facilitate the turnover of focal adhesions as a result of diminished contact with the cytoskeleton.…”

Section: Discussionmentioning

confidence: 99%

Roles of Rho-associated Kinase and Myosin Light Chain Kinase in Morphological and Migratory Defects of Focal Adhesion Kinase-null Cells

Chen

Tzen

Bresnick

et al. 2002

Journal of Biological Chemistry

140

121

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Rho GTPases including Rho, Rac, and Cdc42 are key modulators of the actin cytoskeleton (1-3). They are critical for the cell shape changes and adhesion dynamics that drive cell migration (4 -7). Among the Rho GTPase family, Rho induces the formation of focal adhesions and stress fibers (7,8). Interestingly, although a basal level of Rho activity is needed for fibroblast migration, too much Rho activity impedes migration (4, 9, 10). It has been shown that the concerted action of two of the immediate Rho targets, Rho-associated kinase (Rho-kinase) 1 /ROCK and the formin homology protein mDia1, mediate the effect of Rho on matrix adhesion and the actin cytoskeleton (11). In particular, Rho-kinase was shown to stimulate myosindriven contractility in smooth muscle and nonmuscle cells by phosphorylating, thereby inactivating myosin light chain (MLC) phosphatase (12, 13), and possibly by direct phosphorylation of MLC (14 -16). In addition to Rho-kinase, MLC kinase (MLCK) is another kinase that phosphorylates the MLC in both smooth muscle and nonmuscle cells (17, 18). The phosphorylation of MLC on Ser-19 and to a lesser extent on Thr-18 by MLCK promotes the assembly of myosin II into filaments and activates its ATPase activity, which stabilizes the actin-myosin interaction and promotes cell contractility (19 -22). Recently, Rho-kinase and MLCK were suggested to play distinct roles in spatial regulation of MLC phosphorylation. Rho-kinase appears to be important for MLC phosphorylation in the center of cells, and MLCK is responsible for phosphorylating MLC at the cell periphery (16). Focal adhesion kinase (FAK), a 125-kDa cytoplasmic tyrosine kinase localized in focal contacts, has been known to play an important role in integrin-mediated cell migration (23). Fibroblasts derived from FAK-null mouse embryos are more rounded and poorly spread than their wild-type counterparts (23). They show an overabundance of focal adhesions, enriched cortical actin filaments at the cell periphery, and a decreased migration rate (23,24). It has been suggested that the increase in peripheral adhesions results from an inhibition of turnover in FAK Ϫ/Ϫ cells (23), which may result from constitutive activation of Rho (24). Because of the known involvement of Rhokinase and MLCK in cell contractility, a major factor controlling cell migration, we hypothesize that abnormal regulation of Rho-kinase and MLCK may underlie the migratory defect of FAK Ϫ/Ϫ cells. EXPERIMENTAL PROCEDURESMaterials-Fetal bovine serum, non-essential amino acids, sodium pyruvate, and 2-mercaptoethanol were purchased from Invitrogen. Y27632, a specific inhibitor of Rho-kinase, was purchased from Calbiochem. The monoclonal anti-MLCK, monoclonal anti-MLC, monoclonal anti-␤-tubulin, bovine MLC, myelin basic protein (MBP), cytochalasin D, and 2,3-butanedione monoxime (BDM) were purchased from SigmaAldrich. The polyclonal anti-Rho-kinase and monoclonal anti-paxillin were purchased from Transduction Laboratories (Lexington, KY). The plasmid pEGFP-N1-MLCK and polyclonal anti-MLCK ...

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“…[38][39][40] FAK may also decrease integrin-cytoskeleton linkages through α-actinin phosphorylation, which reduces its affinity for actin. 41,42 Biochemical experiments performed in COS and PTP1B-null cells suggested that PTP1B could dephosphorylate α-actinin and promote chemotaxis to fibronectin. 43 We postulated that PTP1B dephosphorylates α-actinin in adhesions facilitating its binding to actin (Fig.…”

Section: Ptp1b Regulation Of Cell-matrix Coupling To the Cytoskeletonmentioning

confidence: 99%

Protein tyrosine phosphatase PTP1B in cell adhesion and migration

Arregui

González

Burdisso

et al. 2013

Cell Adhesion & Migration

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C ell migration requires a highly coordinated interplay between specialized plasma membrane adhesion complexes and the cytoskeleton. Protein phosphorylation/dephosphorylation modifications regulate many aspects of the integrin-cytoskeleton interdependence, including their coupling, dynamics, and organization to support cell movement. The endoplasmic reticulumbound protein tyrosine phosphatase PTP1B has been implicated as a regulator of cell adhesion and migration. Recent results from our laboratory shed light on potential mechanisms, such as Src/FAK signaling through Rho GTPases and integrin-cytoskeletal coupling.

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The Cytoskeletal/Non-muscle Isoform of α-Actinin Is Phosphorylated on Its Actin-binding Domain by the Focal Adhesion Kinase

Cited by 136 publications

References 65 publications

Quantification of Gel-separated Proteins and Their Phosphorylation Sites by LC-MS Using Unlabeled Internal Standards

Quantification of Gel-separated Proteins and Their Phosphorylation Sites by LC-MS Using Unlabeled Internal Standards

Roles of Rho-associated Kinase and Myosin Light Chain Kinase in Morphological and Migratory Defects of Focal Adhesion Kinase-null Cells

Protein tyrosine phosphatase PTP1B in cell adhesion and migration

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