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MIEN1, a novel interactor of Annexin A2, promotes tumor cell migration by enhancing AnxA2 cell surface expression.

Kpetemey M, Dasgupta S, Rajendiran S, Das S, Gibbs LD, Shetty P, Gryczynski Z, Vishwanatha JK - Mol. Cancer (2015)

Bottom Line: We found that ITAM-phosphorylation of MIEN1 is significantly impaired in isoprenylation-deficient MIEN1 mutants indicating that prenylation of MIEN1 and membrane association is required for cross-phosphorylation of tyrosine residues.Interestingly, our study identified that ectopic overexpression of MIEN1 significantly enhances Tyr23-phosphorylation on AnxA2, thereby stimulating cell surface translocation of AnxA2 and catalyzing the activation of its proteolytic activity.Our study has now deciphered a novel regulatory network governing the vicious process of breast tumor cell invasion-metastasis, and findings suggest MIEN1-AnxA2 as prospective targets to counter the deadly disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Medical Genetics and Institute for Cancer Research, University of North Texas Health Science Center, 3500 Camp Bowie Blvd., Fort Worth, TX, 76107, USA. lkpeteme@live.unthsc.edu.

ABSTRACT

Background: Migration and invasion enhancer 1 (MIEN1) is a novel gene found to be abundantly expressed in breast tumor tissues and functions as a critical regulator of tumor cell migration and invasion to promote systemic metastases. Previous studies have identified post-translational modifications by isoprenylation at the C-terminal tail of MIEN1 to favor its translocation to the inner leaflet of plasma membrane and its function as a membrane-bound adapter molecule. However, the exact molecular events at the membrane interface activating the MIEN1-driven tumor cell motility are vaguely understood.

Methods: MIEN1 was first studied using in-silico analysis on available RNA sequencing data of human breast tissues and its expression was ascertained in breast cells. We performed several assays including co-immunoprecipitation, wound healing, western blotting and immunofluorescence to decipher the molecular events involved in MIEN1-mediated tumor cell migration.

Results: Clinically, MIEN1 is predominantly overexpressed in Her-2 and luminal B subtypes of breast tumors, and its increased expression correlates with poor disease free survival. Molecular studies identified a phosphorylation-dependent activation signal in the immunoreceptor tyrosine based activation motif (ITAM) of MIEN1 and the phosphorylation-deficient MIEN1-mutants (Y39F/50 F) to regulate filopodia generation, migration and invasion. We found that ITAM-phosphorylation of MIEN1 is significantly impaired in isoprenylation-deficient MIEN1 mutants indicating that prenylation of MIEN1 and membrane association is required for cross-phosphorylation of tyrosine residues. Furthermore, we identified MIEN1 as a novel interactor of Annexin A2 (AnxA2), a Ca(2+) -dependent phospholipid binding protein, which serves as an extracellular proteolytic center regulating plasmin generation. Fluorescence resonance energy transfer (FRET) confirmed that MIEN1 physically interacts with AnxA2 and functional studies revealed that they mutually cooperate to accentuate tumor cell motility. Interestingly, our study identified that ectopic overexpression of MIEN1 significantly enhances Tyr23-phosphorylation on AnxA2, thereby stimulating cell surface translocation of AnxA2 and catalyzing the activation of its proteolytic activity.

Conclusion: Our data show that the presence and interaction of both MIEN1 and AnxA2 in breast tumors are crucial drivers of cell motility. Our study has now deciphered a novel regulatory network governing the vicious process of breast tumor cell invasion-metastasis, and findings suggest MIEN1-AnxA2 as prospective targets to counter the deadly disease.

No MeSH data available.


Related in: MedlinePlus

MIEN1 has a functional immunoreceptor tyrosine based activation motif. a Schematic representation of the ITAM and CAAX-prenylation motif on MIEN1 protein and different site directed mutants used in the current study. Tyrosine residues in ITAM domain were mutated to phenylalanine (F) and cysteine residue of the CAAX-prenylation site was mutated to serine (S). b NIH3T3 cells were transfected with GFP fused MIEN1WT, MIEN1Y39F, MIEN1Y50F and MIEN1Y39/50F constructs followed by immunoprecipitation using GFP antibody. The tyrosine phosphorylation of MIEN1 was detected using a generic phospho-tyrosine antibody. c NIH3T3 cells were transfected with empty GFP, MIEN1WT and MIEN1C112S constructs. MIEN1WT transfected cells were either treated with DMSO (vehicle control) or geranylgeranyl transferase I (GGTI) inhibitor and then immunoprecipitated using GFP antibody followed by immunoblotting with phospho-tyrosine antibody. The total MIEN1 protein used for immunoprecipitation was indicated as input
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Fig2: MIEN1 has a functional immunoreceptor tyrosine based activation motif. a Schematic representation of the ITAM and CAAX-prenylation motif on MIEN1 protein and different site directed mutants used in the current study. Tyrosine residues in ITAM domain were mutated to phenylalanine (F) and cysteine residue of the CAAX-prenylation site was mutated to serine (S). b NIH3T3 cells were transfected with GFP fused MIEN1WT, MIEN1Y39F, MIEN1Y50F and MIEN1Y39/50F constructs followed by immunoprecipitation using GFP antibody. The tyrosine phosphorylation of MIEN1 was detected using a generic phospho-tyrosine antibody. c NIH3T3 cells were transfected with empty GFP, MIEN1WT and MIEN1C112S constructs. MIEN1WT transfected cells were either treated with DMSO (vehicle control) or geranylgeranyl transferase I (GGTI) inhibitor and then immunoprecipitated using GFP antibody followed by immunoblotting with phospho-tyrosine antibody. The total MIEN1 protein used for immunoprecipitation was indicated as input

Mentions: In addition to isoprenylation motif [6], MIEN1 harbors several potential phosphorylation sites. Analysis of MIEN1 sequence using computational algorithms (KinasePhos 2.0) identified four potential phosphorylated tyrosine residues (Tyr29, Tyr39, Tyr50 and Tyr85) [22–25] of which Tyr39 and Tyr50 residues are located in the ITAM domain [21]. The canonical ITAM (immunoreceptor tyrosine based activation motif) is an 18 sequence amino acids (YxxI(6–8)YxxL) where tyrosine is separated from a leucine or isoleucine by any two other amino acids, giving the signature YxxL/I; and these two signatures are typically separated by 6 to 8 amino acids. To demonstrate that the Y39/Y50 in the ITAM domain of MIEN1 is phosphorylated, we investigated the phosphorylation status of MIEN1 (Fig. 2a). Immunoprecipitation of GFP-tagged MIEN1 constructs followed by immunoblot analysis using a generic phospho-tyrosine antibody, demonstrated that MIEN1 wild type (MIEN1WT) is tyrosine phosphorylated, whereas the Y39F and Y50F phospho-deficient mutants showed lower phosphorylation status. However Y50F mutant showed a greater loss of phosphorylation compared to Y39F (Fig. 2b). Replacement of Y39 and Y50 with phenylalanine (Y39/Y50F) reduced MIEN1-tyrosine phosphorylation by half; indicating that there is some background phosphorylation on the tyrosine residues outside the ITAM domain.Fig. 2


MIEN1, a novel interactor of Annexin A2, promotes tumor cell migration by enhancing AnxA2 cell surface expression.

Kpetemey M, Dasgupta S, Rajendiran S, Das S, Gibbs LD, Shetty P, Gryczynski Z, Vishwanatha JK - Mol. Cancer (2015)

MIEN1 has a functional immunoreceptor tyrosine based activation motif. a Schematic representation of the ITAM and CAAX-prenylation motif on MIEN1 protein and different site directed mutants used in the current study. Tyrosine residues in ITAM domain were mutated to phenylalanine (F) and cysteine residue of the CAAX-prenylation site was mutated to serine (S). b NIH3T3 cells were transfected with GFP fused MIEN1WT, MIEN1Y39F, MIEN1Y50F and MIEN1Y39/50F constructs followed by immunoprecipitation using GFP antibody. The tyrosine phosphorylation of MIEN1 was detected using a generic phospho-tyrosine antibody. c NIH3T3 cells were transfected with empty GFP, MIEN1WT and MIEN1C112S constructs. MIEN1WT transfected cells were either treated with DMSO (vehicle control) or geranylgeranyl transferase I (GGTI) inhibitor and then immunoprecipitated using GFP antibody followed by immunoblotting with phospho-tyrosine antibody. The total MIEN1 protein used for immunoprecipitation was indicated as input
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Fig2: MIEN1 has a functional immunoreceptor tyrosine based activation motif. a Schematic representation of the ITAM and CAAX-prenylation motif on MIEN1 protein and different site directed mutants used in the current study. Tyrosine residues in ITAM domain were mutated to phenylalanine (F) and cysteine residue of the CAAX-prenylation site was mutated to serine (S). b NIH3T3 cells were transfected with GFP fused MIEN1WT, MIEN1Y39F, MIEN1Y50F and MIEN1Y39/50F constructs followed by immunoprecipitation using GFP antibody. The tyrosine phosphorylation of MIEN1 was detected using a generic phospho-tyrosine antibody. c NIH3T3 cells were transfected with empty GFP, MIEN1WT and MIEN1C112S constructs. MIEN1WT transfected cells were either treated with DMSO (vehicle control) or geranylgeranyl transferase I (GGTI) inhibitor and then immunoprecipitated using GFP antibody followed by immunoblotting with phospho-tyrosine antibody. The total MIEN1 protein used for immunoprecipitation was indicated as input
Mentions: In addition to isoprenylation motif [6], MIEN1 harbors several potential phosphorylation sites. Analysis of MIEN1 sequence using computational algorithms (KinasePhos 2.0) identified four potential phosphorylated tyrosine residues (Tyr29, Tyr39, Tyr50 and Tyr85) [22–25] of which Tyr39 and Tyr50 residues are located in the ITAM domain [21]. The canonical ITAM (immunoreceptor tyrosine based activation motif) is an 18 sequence amino acids (YxxI(6–8)YxxL) where tyrosine is separated from a leucine or isoleucine by any two other amino acids, giving the signature YxxL/I; and these two signatures are typically separated by 6 to 8 amino acids. To demonstrate that the Y39/Y50 in the ITAM domain of MIEN1 is phosphorylated, we investigated the phosphorylation status of MIEN1 (Fig. 2a). Immunoprecipitation of GFP-tagged MIEN1 constructs followed by immunoblot analysis using a generic phospho-tyrosine antibody, demonstrated that MIEN1 wild type (MIEN1WT) is tyrosine phosphorylated, whereas the Y39F and Y50F phospho-deficient mutants showed lower phosphorylation status. However Y50F mutant showed a greater loss of phosphorylation compared to Y39F (Fig. 2b). Replacement of Y39 and Y50 with phenylalanine (Y39/Y50F) reduced MIEN1-tyrosine phosphorylation by half; indicating that there is some background phosphorylation on the tyrosine residues outside the ITAM domain.Fig. 2

Bottom Line: We found that ITAM-phosphorylation of MIEN1 is significantly impaired in isoprenylation-deficient MIEN1 mutants indicating that prenylation of MIEN1 and membrane association is required for cross-phosphorylation of tyrosine residues.Interestingly, our study identified that ectopic overexpression of MIEN1 significantly enhances Tyr23-phosphorylation on AnxA2, thereby stimulating cell surface translocation of AnxA2 and catalyzing the activation of its proteolytic activity.Our study has now deciphered a novel regulatory network governing the vicious process of breast tumor cell invasion-metastasis, and findings suggest MIEN1-AnxA2 as prospective targets to counter the deadly disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Medical Genetics and Institute for Cancer Research, University of North Texas Health Science Center, 3500 Camp Bowie Blvd., Fort Worth, TX, 76107, USA. lkpeteme@live.unthsc.edu.

ABSTRACT

Background: Migration and invasion enhancer 1 (MIEN1) is a novel gene found to be abundantly expressed in breast tumor tissues and functions as a critical regulator of tumor cell migration and invasion to promote systemic metastases. Previous studies have identified post-translational modifications by isoprenylation at the C-terminal tail of MIEN1 to favor its translocation to the inner leaflet of plasma membrane and its function as a membrane-bound adapter molecule. However, the exact molecular events at the membrane interface activating the MIEN1-driven tumor cell motility are vaguely understood.

Methods: MIEN1 was first studied using in-silico analysis on available RNA sequencing data of human breast tissues and its expression was ascertained in breast cells. We performed several assays including co-immunoprecipitation, wound healing, western blotting and immunofluorescence to decipher the molecular events involved in MIEN1-mediated tumor cell migration.

Results: Clinically, MIEN1 is predominantly overexpressed in Her-2 and luminal B subtypes of breast tumors, and its increased expression correlates with poor disease free survival. Molecular studies identified a phosphorylation-dependent activation signal in the immunoreceptor tyrosine based activation motif (ITAM) of MIEN1 and the phosphorylation-deficient MIEN1-mutants (Y39F/50 F) to regulate filopodia generation, migration and invasion. We found that ITAM-phosphorylation of MIEN1 is significantly impaired in isoprenylation-deficient MIEN1 mutants indicating that prenylation of MIEN1 and membrane association is required for cross-phosphorylation of tyrosine residues. Furthermore, we identified MIEN1 as a novel interactor of Annexin A2 (AnxA2), a Ca(2+) -dependent phospholipid binding protein, which serves as an extracellular proteolytic center regulating plasmin generation. Fluorescence resonance energy transfer (FRET) confirmed that MIEN1 physically interacts with AnxA2 and functional studies revealed that they mutually cooperate to accentuate tumor cell motility. Interestingly, our study identified that ectopic overexpression of MIEN1 significantly enhances Tyr23-phosphorylation on AnxA2, thereby stimulating cell surface translocation of AnxA2 and catalyzing the activation of its proteolytic activity.

Conclusion: Our data show that the presence and interaction of both MIEN1 and AnxA2 in breast tumors are crucial drivers of cell motility. Our study has now deciphered a novel regulatory network governing the vicious process of breast tumor cell invasion-metastasis, and findings suggest MIEN1-AnxA2 as prospective targets to counter the deadly disease.

No MeSH data available.


Related in: MedlinePlus