SHP2 Mediates the Localized Activation of Fyn Downstream of the α6β4 Integrin To Promote Carcinoma Invasion

ABSTRACT Src family kinase (SFK) activity is elevated in many cancers, and this activity correlates with aggressive tumor behavior. The α6β4 integrin, which is also associated with a poor prognosis in many tumor types, can stimulate SFK activation; however, the mechanism by which it does so is not known. In the current study, we provide novel mechanistic insight into how the α6β4 integrin selectively activates the Src family member Fyn in response to receptor engagement. Both catalytic and noncatalytic functions of SHP2 are required for Fyn activation by α6β4. Specifically, the tyrosine phosphatase SHP2 is recruited to α6β4 and its catalytic activity is stimulated through a specific interaction of its N-terminal SH2 domain with pY1494 in the β4 subunit. Fyn is recruited to the α6β4/SHP2 complex through an interaction with phospho-Y580 in the C terminus of SHP2. In addition to activating Fyn, this interaction with Y580-SHP2 localizes Fyn to sites of receptor engagement, which is required for α6β4-dependent invasion. Of significance for tumor progression, phosphorylation of Y580-SHP2 and SFK activation are increased in orthotopic human breast tumors that express α6β4 and activation of this pathway is dependent upon Y1494.

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EYA1's Conformation Specificity in Dephosphorylating Phosphothreonine in Myc and Its Activity on Myc Stabilization in Breast Cancer

Molecular and Cellular Biology ◽

10.1128/mcb.00499-16 ◽

2016 ◽

Vol 37 (1) ◽

Cited By ~ 13

Author(s):

Jun Li ◽

Yoel Rodriguez ◽

Chunming Cheng ◽

Lei Zeng ◽

Elaine Y. M. Wong ◽

...

Keyword(s):

Breast Cancer ◽

Human Breast Cancer ◽

Cell Cycle Progression ◽

Tyrosine Phosphatase ◽

Human Breast ◽

Hairpin Rna ◽

Critical Function ◽

Cycle Progression ◽

Haloacid Dehalogenase ◽

Insight Into

ABSTRACT EYA1 is known to be overexpressed in human breast cancer, in which the Myc protein is also accumulated in association with decreased phospho-T58 (pT58) levels. We have recently reported that EYA1 functions as a unique protein phosphatase to dephosphorylate Myc at pT58 to regulate Myc levels. However, it remains unclear whether EYA1-mediated Myc dephosphorylation on T58 is a critical function in regulating Myc protein stability in breast cancer. Furthermore, EYA1's substrate specificity has remained elusive. In this study, we have investigated these questions, and here, we report that depletion of EYA1 using short hairpin RNA (shRNA) in breast cancer cells destabilizes the Myc protein and increases pT58 levels, leading to an increase in the doubling time and impairment of cell cycle progression. In correlation with EYA1-mediated stabilization of cMyc and reduced levels of pT58, EYA1 greatly reduced cMyc-FBW7 binding and cMyc ubiquitination, thus providing novel insight into how EYA1 acts to regulate the FBW7-mediated Myc degradation machinery. We found that the conserved C-terminal haloacid dehalogenase domain of EYA1, which has been reported to have only tyrosine phosphatase activity, has dual phosphatase activities, and both the N- and C-terminal domains interact with substrates to increase the catalytic activity of EYA1. Enzymatic assay and nuclear magnetic resonance (NMR) analysis demonstrated that EYA1 has a striking conformation preference for phospho-T58 of Myc. Together, our results not only provide novel structural evidence about the conformation specificity of EYA1 in dephosphorylating phosphothreonine in Myc but also reveal an important mechanism contributing to Myc deregulation in human breast cancer.

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An allosteric interaction controls the activation mechanism of SHP2 tyrosine phosphatase

Scientific Reports ◽

10.1038/s41598-020-75409-7 ◽

2020 ◽

Vol 10 (1) ◽

Cited By ~ 1

Author(s):

Massimiliano Anselmi ◽

Jochen S. Hub

Keyword(s):

Tyrosine Phosphatase ◽

Binding Mode ◽

Sh2 Domain ◽

Activation Mechanism ◽

Ligand Affinity ◽

Allosteric Interaction ◽

Ligand Stimulation ◽

Novel Strategy ◽

Multiple Signaling ◽

Insight Into

Abstract SHP2 is a protein tyrosine phosphatase (PTP) involved in multiple signaling pathways. Mutations of SHP2 can result in Noonan syndrome or pediatric malignancies. Inhibition of wild-type SHP2 represents a novel strategy against several cancers. SHP2 is activated by binding of a phosphopeptide to the N-SH2 domain of SHP2, thereby favoring dissociation of the N-SH2 domain and exposing the active site on the PTP domain. The conformational transitions controlling ligand affinity and PTP dissociation remain poorly understood. Using molecular simulations, we revealed an allosteric interaction restraining the N-SH2 domain into a SHP2-activating and a stabilizing state. Only ligands selecting for the activating N-SH2 conformation, depending on ligand sequence and binding mode, are effective activators. We validate the model of SHP2 activation by rationalizing modified basal activity and responsiveness to ligand stimulation of several N-SH2 variants. This study provides mechanistic insight into SHP2 activation and may open routes for SHP2 regulation.

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The N Terminus of the Transmembrane Protein BP180 Interacts with the N-terminal Domain of BP230, Thereby Mediating Keratin Cytoskeleton Anchorage to the Cell Surface at the Site of the Hemidesmosome

Molecular Biology of the Cell ◽

10.1091/mbc.11.1.277 ◽

2000 ◽

Vol 11 (1) ◽

pp. 277-286 ◽

Cited By ~ 81

Author(s):

Susan B. Hopkinson ◽

Jonathan C. R. Jones

Keyword(s):

Cell Surface ◽

Bullous Pemphigoid ◽

Transmembrane Protein ◽

Terminal Fragment ◽

C Terminus ◽

N Terminus ◽

Α6β4 Integrin ◽

Two Hybrid ◽

Insight Into

In epidermal cells, the keratin cytoskeleton interacts with the elements in the basement membrane via a multimolecular junction called the hemidesmosome. A major component of the hemidesmosome plaque is the 230-kDa bullous pemphigoid autoantigen (BP230/BPAG1), which connects directly to the keratin-containing intermediate filaments of the cytoskeleton via its C terminus. A second bullous pemphigoid antigen of 180 kDa (BP180/BPAG2) is a type II transmembrane component of the hemidesmosome. Using yeast two-hybrid technology and recombinant proteins, we show that an N-terminal fragment of BP230 can bind directly to an N-terminal fragment of BP180. We have also explored the consequences of expression of the BP230 N terminus in 804G cells that assemble hemidesmosomes in vitro. Unexpectedly, this fragment disrupts the distribution of BP180 in transfected cells but has no apparent impact on the organization of endogenous BP230 and α6β4 integrin. We propose that the BP230 N terminus competes with endogenous BP230 protein for BP180 binding and inhibits incorporation of BP180 into the cell surface at the site of the hemidesmosome. These data provide new insight into those interactions of the molecules of the hemidesmosome that are necessary for its function in integrating epithelial and connective tissue types.

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An allosteric interaction controls the activation mechanism of SHP2 tyrosine phosphatase

10.1101/2020.03.24.004911 ◽

2020 ◽

Author(s):

Massimiliano Anselmi ◽

Jochen S. Hub

Keyword(s):

Tyrosine Phosphatase ◽

Binding Mode ◽

Sh2 Domain ◽

Activation Mechanism ◽

Ligand Affinity ◽

Allosteric Interaction ◽

Ligand Stimulation ◽

Novel Strategy ◽

Multiple Signaling ◽

Insight Into

ABSTRACTSHP2 is a protein tyrosine phosphatase (PTP) involved in multiple signaling pathways. Mutations of SHP2 can result in Noonan syndrome or pediatric malignancies. Inhibition of wild-type SHP2 represents a novel strategy against several cancers. SHP2 is activated by binding of a phosphopeptide to the N-SH2 domain of SHP2, thereby favoring dissociation of the N-SH2 domain and exposing the active site on the PTP domain. The conformational transitions controlling ligand affinity and PTP dissociation remain poorly understood. Using molecular simulations, we revealed an allosteric interaction restraining the N-SH2 domain into a SHP2-activating and a stabilizing state. Only ligands selecting for the activating N-SH2 conformation, depending on ligand sequence and binding mode, are effective activators. We validate the model of SHP2 activation by rationalizing modified basal activity and responsiveness to ligand stimulation of several N-SH2 variants. This study provides mechanistic insight into SHP2 activation and may open routes for SHP2 regulation.

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