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Myosin-interacting guanine exchange factor (MyoGEF) regulates the invasion activity of MDA-MB-231 breast cancer cells through activation of RhoA and RhoC.

Wu D, Asiedu M, Wei Q - Oncogene (2009)

Bottom Line: RNA-interference (RNAi)-mediated depletion of MyoGEF in MDA-MB-231 cells not only suppresses the activation of RhoA and RhoC, but also decreases cell polarity and invasion activity.The dominant-negative mutants of RhoA and RhoC, but not Rac1 and Cdc42, dramatically decrease actin polymerization induced by MyoGEF.Moreover, MyoGEFprotein levels significantly increase in infiltrating ductal carcinomas as well as in invasive breast cancer cell lines.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Kansas State University, Manhattan, KS 66506, USA.

ABSTRACT
The small guanine triphosphatase (GTPase) proteins RhoA and RhoC are essential for tumor invasion and/or metastasis in breast carcinomas. However, it is poorly understood how RhoA and RhoC are activated in breast cancer cells. Here we describe the role of myosin-interacting guanine nucleotide exchange factor (Myo-GEF) in regulating RhoA and RhoC activation as well as cell polarity and invasion in an invasive breast cancer cell line MDA-MB-231. RNA-interference (RNAi)-mediated depletion of MyoGEF in MDA-MB-231 cells not only suppresses the activation of RhoA and RhoC, but also decreases cell polarity and invasion activity. The dominant-negative mutants of RhoA and RhoC, but not Rac1 and Cdc42, dramatically decrease actin polymerization induced by MyoGEF. In addition, MyoGEF co-localizes with nonmuscle myosin IIA (NMIIA) to the front of migrating cells, and depletion of NMIIA by RNAi disrupts the polarized localization of MyoGEF at the cell leading edge, suggesting a role for NMIIA in regulating MyoGEF localization and function. Moreover, MyoGEFprotein levels significantly increase in infiltrating ductal carcinomas as well as in invasive breast cancer cell lines. Taken together, our results suggest that MyoGEF cooperates with NMIIA to regulate the polarity and invasion activity of breast cancer cells through activation of RhoA and RhoC.

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MyoGEF interacts with NMIIA(A) MDA-MB-231 cells expressing Myc-MyoGEF were subjected to immunoprecipitation with anti-Myc antibody followed by immunoblot analysis with anti-IIA or anti-IIB antibodies. Note that Myc-MyoGEF binds to NMIIA but not NMIIB. (B) Schematic diagram of MyoGEF fragments that were used in (C). (C) Interactions between Myc-tagged MyoGEF fragments and endogenous NMIIA. Full-length MyoGEF (lane 3) as well as MyoGEF fragments Myc-PH (lane 5), Myc-1-409 (lane 8), and Myc-1-500 (lane 9) could pull down a significant amount of endogenous NMIIA. Note that cell lysate from lane 3 was also used for immunoprecipitation with normal IgG (lane 2). ~5% of cell lysates were loaded.
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Figure 5: MyoGEF interacts with NMIIA(A) MDA-MB-231 cells expressing Myc-MyoGEF were subjected to immunoprecipitation with anti-Myc antibody followed by immunoblot analysis with anti-IIA or anti-IIB antibodies. Note that Myc-MyoGEF binds to NMIIA but not NMIIB. (B) Schematic diagram of MyoGEF fragments that were used in (C). (C) Interactions between Myc-tagged MyoGEF fragments and endogenous NMIIA. Full-length MyoGEF (lane 3) as well as MyoGEF fragments Myc-PH (lane 5), Myc-1-409 (lane 8), and Myc-1-500 (lane 9) could pull down a significant amount of endogenous NMIIA. Note that cell lysate from lane 3 was also used for immunoprecipitation with normal IgG (lane 2). ~5% of cell lysates were loaded.

Mentions: We previously showed that MyoGEF interacts with NMIIA in vitro (Wu et al., 2006). Three isoforms of the nonmuscle myosin heavy chain (NMHC), IIA, IIB, and IIC, have been identified in humans and mice (Bresnick, 1999; Conti and Adelstein, 2008; Golomb et al., 2004; Krendel and Mooseker, 2005; Sellers, 2000). MDA-MB-231 cells express IIA and IIB, but not IIC (Betapudi et al., 2006). To confirm the interaction between MyoGEF and NMII, MDA-MB-231 cells expressing Myc-MyoGEF were subjected to immunoprecipitation with anti-Myc antibody followed by immunoblot analysis with anti-IIA or anti-IIB antibodies. As shown in Figure 5A, endogenous NMIIA, but not NMIIB, could be co-immunoprecipitated with Myc-MyoGEF from total cell lysates of transfected MDA-MB-231 cells, indicating that MyoGEF can preferentially interact with NMIIA in MDA-MB-231 cells. To further characterize the interaction between MyoGEF and NMIIA, we generated different truncated versions of MyoGEF (Figure 5B). Plasmids encoding these Myc-tagged MyoGEF fragments were transfected into a HeLa cell line that only expresses NMIIA, but not NMIIB and NMIIC (Wei and Adelstein, 2000). As shown in Figure 5C, Myc-MyoGEF full-length, Myc-PH, Myc-1-409, and Myc-1-500 could immunoprecipitate a significant amount of NMIIA, suggesting that the PH domain as well as the 351-409 region that is C-terminal to the DH domain are required for interaction with NMIIA.


Myosin-interacting guanine exchange factor (MyoGEF) regulates the invasion activity of MDA-MB-231 breast cancer cells through activation of RhoA and RhoC.

Wu D, Asiedu M, Wei Q - Oncogene (2009)

MyoGEF interacts with NMIIA(A) MDA-MB-231 cells expressing Myc-MyoGEF were subjected to immunoprecipitation with anti-Myc antibody followed by immunoblot analysis with anti-IIA or anti-IIB antibodies. Note that Myc-MyoGEF binds to NMIIA but not NMIIB. (B) Schematic diagram of MyoGEF fragments that were used in (C). (C) Interactions between Myc-tagged MyoGEF fragments and endogenous NMIIA. Full-length MyoGEF (lane 3) as well as MyoGEF fragments Myc-PH (lane 5), Myc-1-409 (lane 8), and Myc-1-500 (lane 9) could pull down a significant amount of endogenous NMIIA. Note that cell lysate from lane 3 was also used for immunoprecipitation with normal IgG (lane 2). ~5% of cell lysates were loaded.
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Related In: Results  -  Collection

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Figure 5: MyoGEF interacts with NMIIA(A) MDA-MB-231 cells expressing Myc-MyoGEF were subjected to immunoprecipitation with anti-Myc antibody followed by immunoblot analysis with anti-IIA or anti-IIB antibodies. Note that Myc-MyoGEF binds to NMIIA but not NMIIB. (B) Schematic diagram of MyoGEF fragments that were used in (C). (C) Interactions between Myc-tagged MyoGEF fragments and endogenous NMIIA. Full-length MyoGEF (lane 3) as well as MyoGEF fragments Myc-PH (lane 5), Myc-1-409 (lane 8), and Myc-1-500 (lane 9) could pull down a significant amount of endogenous NMIIA. Note that cell lysate from lane 3 was also used for immunoprecipitation with normal IgG (lane 2). ~5% of cell lysates were loaded.
Mentions: We previously showed that MyoGEF interacts with NMIIA in vitro (Wu et al., 2006). Three isoforms of the nonmuscle myosin heavy chain (NMHC), IIA, IIB, and IIC, have been identified in humans and mice (Bresnick, 1999; Conti and Adelstein, 2008; Golomb et al., 2004; Krendel and Mooseker, 2005; Sellers, 2000). MDA-MB-231 cells express IIA and IIB, but not IIC (Betapudi et al., 2006). To confirm the interaction between MyoGEF and NMII, MDA-MB-231 cells expressing Myc-MyoGEF were subjected to immunoprecipitation with anti-Myc antibody followed by immunoblot analysis with anti-IIA or anti-IIB antibodies. As shown in Figure 5A, endogenous NMIIA, but not NMIIB, could be co-immunoprecipitated with Myc-MyoGEF from total cell lysates of transfected MDA-MB-231 cells, indicating that MyoGEF can preferentially interact with NMIIA in MDA-MB-231 cells. To further characterize the interaction between MyoGEF and NMIIA, we generated different truncated versions of MyoGEF (Figure 5B). Plasmids encoding these Myc-tagged MyoGEF fragments were transfected into a HeLa cell line that only expresses NMIIA, but not NMIIB and NMIIC (Wei and Adelstein, 2000). As shown in Figure 5C, Myc-MyoGEF full-length, Myc-PH, Myc-1-409, and Myc-1-500 could immunoprecipitate a significant amount of NMIIA, suggesting that the PH domain as well as the 351-409 region that is C-terminal to the DH domain are required for interaction with NMIIA.

Bottom Line: RNA-interference (RNAi)-mediated depletion of MyoGEF in MDA-MB-231 cells not only suppresses the activation of RhoA and RhoC, but also decreases cell polarity and invasion activity.The dominant-negative mutants of RhoA and RhoC, but not Rac1 and Cdc42, dramatically decrease actin polymerization induced by MyoGEF.Moreover, MyoGEFprotein levels significantly increase in infiltrating ductal carcinomas as well as in invasive breast cancer cell lines.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Kansas State University, Manhattan, KS 66506, USA.

ABSTRACT
The small guanine triphosphatase (GTPase) proteins RhoA and RhoC are essential for tumor invasion and/or metastasis in breast carcinomas. However, it is poorly understood how RhoA and RhoC are activated in breast cancer cells. Here we describe the role of myosin-interacting guanine nucleotide exchange factor (Myo-GEF) in regulating RhoA and RhoC activation as well as cell polarity and invasion in an invasive breast cancer cell line MDA-MB-231. RNA-interference (RNAi)-mediated depletion of MyoGEF in MDA-MB-231 cells not only suppresses the activation of RhoA and RhoC, but also decreases cell polarity and invasion activity. The dominant-negative mutants of RhoA and RhoC, but not Rac1 and Cdc42, dramatically decrease actin polymerization induced by MyoGEF. In addition, MyoGEF co-localizes with nonmuscle myosin IIA (NMIIA) to the front of migrating cells, and depletion of NMIIA by RNAi disrupts the polarized localization of MyoGEF at the cell leading edge, suggesting a role for NMIIA in regulating MyoGEF localization and function. Moreover, MyoGEFprotein levels significantly increase in infiltrating ductal carcinomas as well as in invasive breast cancer cell lines. Taken together, our results suggest that MyoGEF cooperates with NMIIA to regulate the polarity and invasion activity of breast cancer cells through activation of RhoA and RhoC.

Show MeSH
Related in: MedlinePlus