Tyrosine Phosphorylation of Caldesmon Is Required for Binding to the Shc·Grb2 Complex

Wang, Ze; Danielsen, Andrew J.; Maihle, Nita J.; McManus, Michael J.

doi:10.1074/jbc.274.47.33807

Cited by 14 publications

(16 citation statements)

References 49 publications

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“…These results were confirmed and extended using site-directed mutants of caldesmon, which individually lack each of the three previously characterized phosphotyrosine acceptor sites of caldesmon (i.e. tyrosines 27, 165, and 363) (Wang et al, 1999). Figure 5 illustrates that only phosphotyrosine-27 of caldesmon is required for binding to either the aminoterminal SH3 or central SH2 domains of Grb2.…”

Section: Grb2 Interacts With Caldesmon Through the Aminoterminal Sh3 supporting

confidence: 61%

“…We have previously shown that transformation by S3-vErbB involves the tyrosine phosphorylation of several novel cytoplasmic targets, and is not simply the amplification of well-characterized mitogenic signaling events (McManus et al, 1995Wang et al, 1999;Boerner et al, 2000Boerner et al, , 2001. In this report, we demonstrate that Grb2 plays a central role in modulating the integrity of the actin-based cytoskeleton during ligand-independent S3-v-ErbB-mediated transformation.…”

Section: Grb2 and Caldesmon Interactions In Transformed Fibroblastsmentioning

confidence: 50%

“…This phosphoprotein complex includes the adapter proteins Shc and Grb2, as well as the cytoskeletal regulatory proteins Pak and caldesmon (McManus et al, 1995Wang et al, 1999).…”

Section: Introductionmentioning

confidence: 99%

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Grb2 regulation of the actin-based cytoskeleton is required for ligand-independent EGF receptor-mediated oncogenesis

et al. 2003

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Mutations within members of the EGF/ErbB receptor family frequently release the oncogenic potential of these receptors, resulting in the activation of downstream signaling events independent of ligand regulatory constraints. We previously have demonstrated that the signal transduction events originating from S3-v-ErbB, a ligandindependent, oncogenic EGF receptor mutant, are qualitatively distinct from the ligand-dependent mitogenic signaling pathways associated with the wild-type EGF receptor. Specifically, expression of S3-v-ErbB in primary fibroblasts results in anchorage-independent growth, increased invasive potential, and the formation of a transformation-specific phosphoprotein signaling complex, all in a Ras-independent manner. Here we demonstrate the transformation-specific interaction between two components of this complex: the adaptor protein Grb2 and the cytoskeletal regulatory protein caldesmon. This interaction is mediated via both the amino-terminal SH3 and central SH2 domains of Grb2, and the aminoterminal (myosin-binding) domain of caldesmon. Expression of a dominant-negative Grb2 deletion mutant, which lacks the carboxy-terminal SH3 domain, in fibroblasts expressing S3-v-ErbB results in a reduction in phosphoprotein complex formation, the loss of anchorageindependent growth, and a reduction in invasive potential.Together, these results demonstrate a Ras-independent role for Grb2 in modulating cytoskeletal function during ligand-independent EGF receptor-mediated transformation, and provide further support for the hypothesis that ligand-independent oncogenic signaling is qualitatively distinct from ligand-dependent mitogenic signaling by the EGF receptor.

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Section: Grb2 Interacts With Caldesmon Through the Aminoterminal Sh3 supporting

confidence: 61%

Section: Grb2 and Caldesmon Interactions In Transformed Fibroblastsmentioning

confidence: 50%

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Grb2 regulation of the actin-based cytoskeleton is required for ligand-independent EGF receptor-mediated oncogenesis

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“…It is clear that the phosphotyrosine protein complex in vErbB-transformed fibroblasts contains several components that potentially can regulate the actomyosin-based cytoskeleton. One such regulatory protein is the tyrosine-phosphorylated form of caldesmon, which is a stable component of a Shc-Grb2 signal adapter complex in v-ErbB-transformed fibroblasts (21). To determine if this Shc-Grb2-caldesmon phosphoprotein complex is a stable subunit of an even larger multiprotein module involving Nck and Pak, we looked for a caldesmon-Pak interaction by co-precipitation assay.…”

Section: Formation Of a Transformation-specific Signaling Complex In mentioning

confidence: 99%

An Oncogenic Epidermal Growth Factor Receptor Signals via a p21-activated Kinase-Caldesmon-Myosin Phosphotyrosine Complex

McManus

Boerner

Danielsen

et al. 2000

Journal of Biological Chemistry

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Many ligand-independent receptor tyrosine kinases are tumorigenic. The biochemical signals that mediate ligand-independent transformation of cells by these transmembrane receptors are poorly defined. In this report, we demonstrate that a constitutively activated mutant epidermal growth factor receptor (v-ErbB) induces the formation of a transformation-specific signaling module that complexes with myosin II. The components of this signaling complex include the signal adapter proteins Shc, Grb2, and Nck, and tyrosine-phosphorylated forms of p21-activated kinase (Pak), caldesmon, and myosin light chain kinase. Transformationspecific, tyrosine phosphorylation of Pak enhances the catalytic activity of this serine/threonine kinase. Furthermore, the tyrosine phosphorylation of Pak is Rho-, but not Ras-, Rac-, or Cdc42-dependent. These results demonstrate that a ligand-independent epidermal growth factor receptor mutant can transduce oncogenic signals that are distinct from ligand-dependent, mitogenic signals. In addition, these data provide evidence for the coupling of oncogenic receptor tyrosine kinases with the actomyosin molecular motor. This myosinassociated signaling module may mediate some of the biochemical changes of myosin found in v-ErbBtransformed fibroblasts, thereby contributing to the regulation of the mechanical forces governing cellular adhesion, cytoskeletal tension, and, hence, anchorageindependent cell growth.Mutations in receptor tyrosine kinases may result in ligandindependent, constitutive kinase activity and tumorigenesis (1-3). The biochemical signals that mediate transformation by these ligand-independent receptors are poorly defined, including the signals that influence the reorganization of the actomyosin-based cytoskeleton and anchorage-independent cell growth. Two fundamentally distinct mechanisms of oncogenic signaling may be at work: (i) the persistent stimulation of mitogenic signaling pathways by the kinase, or (ii) the stimulation of novel, transformation-specific, signaling networks arising from altered substrate specificity of the kinase. To distinguish between these alternative models, we have studied fibroblast transformation by v-ErbB, a ligand-independent, oncogenic homolog of the human epidermal growth factor receptor (EGFR). 1Our previous studies provided compelling evidence that there are qualitatively distinct signaling pathways associated with v-ErbB-mediated transformation compared with liganddependent, mitogenic signaling. Specifically, we identified a novel, transformation-associated phosphotyrosine protein complex (4). This Src homologous collagen protein (Shc)-phosphotyrosine multiprotein complex forms in fibroblasts expressing a transforming v-ErbB protein, but not in cells stimulated with ligand, or in cells expressing a non-transforming v-ErbB mutant with a constitutively active kinase domain (4). In addition to the signal adapter protein Shc, this protein complex includes growth factor receptor binding protein-2 (Grb2), a 78-kilodalton (kDa) tyrosine-phosphorylated ...

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“…In addition, two cytoskeletal associated proteins, i.e. caldesmon and p21-activated kinase, are uniquely tyrosine-phosphorylated in S3v-ErbB-transformed cells (2,4,38). Interestingly, Ras activation, a key aspect of ligand-dependent EGFR mitogenic signaling, is not required for S3v-ErbB-mediated transformation or for these cytoskeletal changes (5).…”

mentioning

confidence: 99%

Activation of Rho Is Required for Ligand-independent Oncogenic Signaling by a Mutant Epidermal Growth Factor Receptor

Boerner

Danielsen

McManus

et al. 2001

Journal of Biological Chemistry

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Mutations in the epidermal growth factor receptor have been identified in several human tumor types, including gliomas. These receptor mutants have deletions in their extracellular ligand-binding domains and are, therefore, no longer regulated by ligand, resulting in constitutive activation of the receptor kinase. These mutants have been proposed to transduce oncogenic signals via ligand-independent signaling pathways. Avian viral homologues of these oncogenic epidermal growth factor receptors exhibit structurally homologous deletions and form tumors in chickens. One such mutant, S3v-ErbB, transforms fibroblasts in vitro, and transformation has been correlated with the formation of a novel tyrosine phosphoprotein complex. V-ErbB-mediated complex formation and transformation have been shown to occur independently of Ras activation. The major aims of this study are to further characterize this ligand-independent v-ErbB oncogenic signaling pathway. Here we show that both vErbB-mediated phosphoprotein complex formation and transformation are inhibited by a dominant negative mutant of Rho. This inhibition is specific for dominant negative Rho; dominant negative mutants of Rac and Cdc42 have no effect on transformation or on tyrosine phosphorylation of the phosphoprotein complex. Based on these observations, we propose that S3v-ErbB stimulates a Rho-dependent tyrosine kinase, resulting in complex formation and ultimately oncogenic transformation.An avian viral mutant of the EGFR, 1 S3v-ErbB, transforms fibroblasts in vitro, and in vivo expression results in the development of fibrosarcomas and hemangiosarcomas (1). S3v-ErbB transformation of fibroblasts results in the loss of anchorage-dependent cell growth. This growth pattern has been correlated with specific cytoskeletal changes in v-ErbBtransformed fibroblasts (2, 38). Specifically, stress fibers are disassembled, and myosin light chain kinase activity is reduced (2, 38). In addition, two cytoskeletal associated proteins, i.e. caldesmon and p21-activated kinase, are uniquely tyrosine-phosphorylated in S3v-ErbB-transformed cells (2,4,38). Interestingly, Ras activation, a key aspect of ligand-dependent EGFR mitogenic signaling, is not required for S3v-ErbB-mediated transformation or for these cytoskeletal changes (5). Together, these studies suggest that ligandindependent oncogenic signaling occurs through a pathway that is distinct from the well characterized ligand-dependent mitogenic signaling pathway of EGFR.Rho, Rac, and Cdc42 are small GTP-binding proteins important in the dynamic reorganization of the cytoskeleton of the cell. Rho is a crucial regulator of such cytoskeletal events and functions by controlling stress fiber assembly and focal adhesion formation (6). In contrast, Rac is important for membrane ruffling, lamellipodia formation, and focal complex formation (7-10), whereas Cdc42 is required for filopodia formation (11). The coordinated interplay of the activities of these proteins results in the control of complex biological events such as cell move...

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Tyrosine Phosphorylation of Caldesmon Is Required for Binding to the Shc·Grb2 Complex

Abstract: S3-v-erbB isTogether, these studies demonstrate that the major sites of tyrosine phosphorylation on CaD are located in the myosin and actin binding domains of CaD and that Tyr-27 is the major tyrosine phosphorylation site through which CaD interacts with the Shc⅐Grb2 complex.

Cited by 14 publications

References 49 publications

Grb2 regulation of the actin-based cytoskeleton is required for ligand-independent EGF receptor-mediated oncogenesis

Grb2 regulation of the actin-based cytoskeleton is required for ligand-independent EGF receptor-mediated oncogenesis

An Oncogenic Epidermal Growth Factor Receptor Signals via a p21-activated Kinase-Caldesmon-Myosin Phosphotyrosine Complex

Activation of Rho Is Required for Ligand-independent Oncogenic Signaling by a Mutant Epidermal Growth Factor Receptor

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