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Collagen I-mediated up-regulation of N-cadherin requires cooperative signals from integrins and discoidin domain receptor 1.

Shintani Y, Fukumoto Y, Chaika N, Svoboda R, Wheelock MJ, Johnson KR - J. Cell Biol. (2008)

Bottom Line: Focal adhesion kinase (FAK)-related protein tyrosine kinase (Pyk2) is downstream of DDR1, whereas FAK is downstream of alpha2beta1 integrin.Both receptor complexes rely on the p130 Crk-associated substrate scaffold.Interestingly, Rap1, but not Rho family guanosine triphosphatases, is required for the response to collagen I.

View Article: PubMed Central - PubMed

Affiliation: Department of Oral Biology, University of Nebraska Medical Center, Omaha, NE 68198, USA.

ABSTRACT
Tumor cells undergo epithelial-to-mesenchymal transition (EMT) to convert from a benign to a malignant phenotype. Our recent focus has been signaling pathways that promote EMT in response to collagen. We have shown that human pancreatic cancer cells respond to collagen by up-regulating N-cadherin, which promotes tumor growth, invasion, and metastasis. Initial characterization showed that knocking down c-Jun NH2-terminal kinase prevented N-cadherin up-regulation and limited tumor growth and invasion in a mouse model for pancreatic cancer. The current study was designed to understand the pathway from collagen to N-cadherin up-regulation. Initiation of the signal requires two collagen receptors, alpha2beta1 integrin and discoidin domain receptor (DDR) 1. Each receptor propagates signals through separate pathways that converge to up-regulate N-cadherin. Focal adhesion kinase (FAK)-related protein tyrosine kinase (Pyk2) is downstream of DDR1, whereas FAK is downstream of alpha2beta1 integrin. Both receptor complexes rely on the p130 Crk-associated substrate scaffold. Interestingly, Rap1, but not Rho family guanosine triphosphatases, is required for the response to collagen I.

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p130CAS plays a key role in collagen I–induced changes in BxPC3 cells. (A) 600 μg of protein from BxPC3 cells 12 h after plating on noncoated or collagen I–coated dishes was immunoprecipitated with rabbit IgG or anti-DDR1 rabbit pAb and blotted for p130CAS or DDR1. (B) 600 μg of protein from BxPC3 cells 12 h after plating on noncoated or collagen I–coated dishes was immunoprecipitated with mouse IgG or anti-p130CAS mouse mAb and blotted for DDR1 or p130CAS. (C) BxPC3 cells expressing the neomycin resistance gene (mock), Src*/CAS(SD) (dominant-negative p130CAS), or SrcKM/CAS(SD) (inactive control for dominant-negative p130CAS) were extracted in RIPA buffer 2 d after plating on noncoated or collagen I–coated dishes. 30 μg of protein was resolved by SDS-PAGE and blotted for N-cadherin, myc tag (to detect p130CAS), or tubulin. (D) Mock BxPC3 cells (a and b), cells expressing Src*/CAS(SD) (c and d), or cells expressing SrcKM/CAS(SD) (e and f) were cultured on noncoated (a, c, and e) or collagen I–coated dishes (b, d, and f) for 2 d. Bar, 100 μm.
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fig5: p130CAS plays a key role in collagen I–induced changes in BxPC3 cells. (A) 600 μg of protein from BxPC3 cells 12 h after plating on noncoated or collagen I–coated dishes was immunoprecipitated with rabbit IgG or anti-DDR1 rabbit pAb and blotted for p130CAS or DDR1. (B) 600 μg of protein from BxPC3 cells 12 h after plating on noncoated or collagen I–coated dishes was immunoprecipitated with mouse IgG or anti-p130CAS mouse mAb and blotted for DDR1 or p130CAS. (C) BxPC3 cells expressing the neomycin resistance gene (mock), Src*/CAS(SD) (dominant-negative p130CAS), or SrcKM/CAS(SD) (inactive control for dominant-negative p130CAS) were extracted in RIPA buffer 2 d after plating on noncoated or collagen I–coated dishes. 30 μg of protein was resolved by SDS-PAGE and blotted for N-cadherin, myc tag (to detect p130CAS), or tubulin. (D) Mock BxPC3 cells (a and b), cells expressing Src*/CAS(SD) (c and d), or cells expressing SrcKM/CAS(SD) (e and f) were cultured on noncoated (a, c, and e) or collagen I–coated dishes (b, d, and f) for 2 d. Bar, 100 μm.

Mentions: The next objective of this study was to determine how the signal gets from FAK/Pyk2 to JNK. Signaling cascades often involve scaffolds, and p130CAS plays a crucial role in controlling integrin-dependent processes (Chodniewicz and Klemke, 2004). In addition, the SH3 domain of p130CAS binds FAK and Pyk2 leading to activation of JNK (Tanaka and Hanafusa, 1998; Blaukat et al., 1999). Thus, we asked if DDR1 was also capable of binding to p130CAS. Coimmunoprecipitation showed that DDR1 was in a complex with p130CAS, and this association was increased when cells were plated on collagen I (Fig. 5, A and B).


Collagen I-mediated up-regulation of N-cadherin requires cooperative signals from integrins and discoidin domain receptor 1.

Shintani Y, Fukumoto Y, Chaika N, Svoboda R, Wheelock MJ, Johnson KR - J. Cell Biol. (2008)

p130CAS plays a key role in collagen I–induced changes in BxPC3 cells. (A) 600 μg of protein from BxPC3 cells 12 h after plating on noncoated or collagen I–coated dishes was immunoprecipitated with rabbit IgG or anti-DDR1 rabbit pAb and blotted for p130CAS or DDR1. (B) 600 μg of protein from BxPC3 cells 12 h after plating on noncoated or collagen I–coated dishes was immunoprecipitated with mouse IgG or anti-p130CAS mouse mAb and blotted for DDR1 or p130CAS. (C) BxPC3 cells expressing the neomycin resistance gene (mock), Src*/CAS(SD) (dominant-negative p130CAS), or SrcKM/CAS(SD) (inactive control for dominant-negative p130CAS) were extracted in RIPA buffer 2 d after plating on noncoated or collagen I–coated dishes. 30 μg of protein was resolved by SDS-PAGE and blotted for N-cadherin, myc tag (to detect p130CAS), or tubulin. (D) Mock BxPC3 cells (a and b), cells expressing Src*/CAS(SD) (c and d), or cells expressing SrcKM/CAS(SD) (e and f) were cultured on noncoated (a, c, and e) or collagen I–coated dishes (b, d, and f) for 2 d. Bar, 100 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2290851&req=5

fig5: p130CAS plays a key role in collagen I–induced changes in BxPC3 cells. (A) 600 μg of protein from BxPC3 cells 12 h after plating on noncoated or collagen I–coated dishes was immunoprecipitated with rabbit IgG or anti-DDR1 rabbit pAb and blotted for p130CAS or DDR1. (B) 600 μg of protein from BxPC3 cells 12 h after plating on noncoated or collagen I–coated dishes was immunoprecipitated with mouse IgG or anti-p130CAS mouse mAb and blotted for DDR1 or p130CAS. (C) BxPC3 cells expressing the neomycin resistance gene (mock), Src*/CAS(SD) (dominant-negative p130CAS), or SrcKM/CAS(SD) (inactive control for dominant-negative p130CAS) were extracted in RIPA buffer 2 d after plating on noncoated or collagen I–coated dishes. 30 μg of protein was resolved by SDS-PAGE and blotted for N-cadherin, myc tag (to detect p130CAS), or tubulin. (D) Mock BxPC3 cells (a and b), cells expressing Src*/CAS(SD) (c and d), or cells expressing SrcKM/CAS(SD) (e and f) were cultured on noncoated (a, c, and e) or collagen I–coated dishes (b, d, and f) for 2 d. Bar, 100 μm.
Mentions: The next objective of this study was to determine how the signal gets from FAK/Pyk2 to JNK. Signaling cascades often involve scaffolds, and p130CAS plays a crucial role in controlling integrin-dependent processes (Chodniewicz and Klemke, 2004). In addition, the SH3 domain of p130CAS binds FAK and Pyk2 leading to activation of JNK (Tanaka and Hanafusa, 1998; Blaukat et al., 1999). Thus, we asked if DDR1 was also capable of binding to p130CAS. Coimmunoprecipitation showed that DDR1 was in a complex with p130CAS, and this association was increased when cells were plated on collagen I (Fig. 5, A and B).

Bottom Line: Focal adhesion kinase (FAK)-related protein tyrosine kinase (Pyk2) is downstream of DDR1, whereas FAK is downstream of alpha2beta1 integrin.Both receptor complexes rely on the p130 Crk-associated substrate scaffold.Interestingly, Rap1, but not Rho family guanosine triphosphatases, is required for the response to collagen I.

View Article: PubMed Central - PubMed

Affiliation: Department of Oral Biology, University of Nebraska Medical Center, Omaha, NE 68198, USA.

ABSTRACT
Tumor cells undergo epithelial-to-mesenchymal transition (EMT) to convert from a benign to a malignant phenotype. Our recent focus has been signaling pathways that promote EMT in response to collagen. We have shown that human pancreatic cancer cells respond to collagen by up-regulating N-cadherin, which promotes tumor growth, invasion, and metastasis. Initial characterization showed that knocking down c-Jun NH2-terminal kinase prevented N-cadherin up-regulation and limited tumor growth and invasion in a mouse model for pancreatic cancer. The current study was designed to understand the pathway from collagen to N-cadherin up-regulation. Initiation of the signal requires two collagen receptors, alpha2beta1 integrin and discoidin domain receptor (DDR) 1. Each receptor propagates signals through separate pathways that converge to up-regulate N-cadherin. Focal adhesion kinase (FAK)-related protein tyrosine kinase (Pyk2) is downstream of DDR1, whereas FAK is downstream of alpha2beta1 integrin. Both receptor complexes rely on the p130 Crk-associated substrate scaffold. Interestingly, Rap1, but not Rho family guanosine triphosphatases, is required for the response to collagen I.

Show MeSH
Related in: MedlinePlus