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Palmitoylation supports assembly and function of integrin-tetraspanin complexes.

Yang X, Kovalenko OV, Tang W, Claas C, Stipp CS, Hemler ME - J. Cell Biol. (2004)

Bottom Line: There is also a functional connection between CD9 and beta4 integrins, as evidenced by anti-CD9 antibody effects on beta4-dependent cell spreading.Notably, beta4 palmitoylation neither increased localization into "light membrane" fractions of sucrose gradients nor decreased solubility in nonionic detergents-hence it does not promote lipid raft association.Instead, palmitoylation of beta4 (and of the closely associated tetraspanin CD151) promotes CD151-alpha6beta4 incorporation into a network of secondary tetraspanin interactions (with CD9, CD81, CD63, etc.), which provides a novel framework for functional regulation.

View Article: PubMed Central - PubMed

Affiliation: Dana-Farber Cancer Institute and Department of Pathology, Harvard Medical School, Boston, MA 02115, USA.

ABSTRACT
As observed previously, tetraspanin palmitoylation promotes tetraspanin microdomain assembly. Here, we show that palmitoylated integrins (alpha3, alpha6, and beta4 subunits) and tetraspanins (CD9, CD81, and CD63) coexist in substantially overlapping complexes. Removal of beta4 palmitoylation sites markedly impaired cell spreading and signaling through p130Cas on laminin substrate. Also in palmitoylation-deficient beta4, secondary associations with tetraspanins (CD9, CD81, and CD63) were diminished and cell surface CD9 clustering was decreased, whereas core alpha6beta4-CD151 complex formation was unaltered. There is also a functional connection between CD9 and beta4 integrins, as evidenced by anti-CD9 antibody effects on beta4-dependent cell spreading. Notably, beta4 palmitoylation neither increased localization into "light membrane" fractions of sucrose gradients nor decreased solubility in nonionic detergents-hence it does not promote lipid raft association. Instead, palmitoylation of beta4 (and of the closely associated tetraspanin CD151) promotes CD151-alpha6beta4 incorporation into a network of secondary tetraspanin interactions (with CD9, CD81, CD63, etc.), which provides a novel framework for functional regulation.

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β4 integrin–mediated cell spreading. (A) MDA-MB-435 cells stably expressing vector or wild-type (WT) or 7C/S β4 were held in suspension at 37°C for 30 min, and then were plated on wells coated in PBS at 4°C overnight with either laminin-5 (LN; subpanels A–C) or vitronectin (VN; subpanels D–E), and photographed after 45 min. Cell spreading was monitored using a microscope (Axiovert 135; Carl Zeiss MicroImaging, Inc.) as described previously (Stipp and Hemler, 2000). Bar, 50 μm. (B) Cell spreading was estimated by determining the percentage of cells that were no longer round and phase-bright. The accuracy of this method was validated by quantitation of cell area using Scion Image software, which confirmed that we could readily distinguish cells in which the area had increased by ≥1.2-fold. Results (mean ± SD) are derived from at least two separate experiments, counting at least two representative fields in each experiment, with at least 50 cells/field. (C) MDA-MB-435 cells were suspended at 37°C for 30 min, and then either retained in suspension or plated on vitronectin (Vn), laminin-1, or laminin-5 for 45 min. Some samples (lanes 8–10) were preincubated with 1.0 μg/ml PP2 in DMSO for 20 min before plating. Cells were collected and lysed in RIPA, and p130Cas was immunoprecipitated. After SDS-PAGE, samples were blotted with antiphosphotyrosine mAb 4G10. To assess protein loading, the blot was stripped and reblotted with p130Cas-specific antibody. Similar results were seen in multiple experiments.
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fig5: β4 integrin–mediated cell spreading. (A) MDA-MB-435 cells stably expressing vector or wild-type (WT) or 7C/S β4 were held in suspension at 37°C for 30 min, and then were plated on wells coated in PBS at 4°C overnight with either laminin-5 (LN; subpanels A–C) or vitronectin (VN; subpanels D–E), and photographed after 45 min. Cell spreading was monitored using a microscope (Axiovert 135; Carl Zeiss MicroImaging, Inc.) as described previously (Stipp and Hemler, 2000). Bar, 50 μm. (B) Cell spreading was estimated by determining the percentage of cells that were no longer round and phase-bright. The accuracy of this method was validated by quantitation of cell area using Scion Image software, which confirmed that we could readily distinguish cells in which the area had increased by ≥1.2-fold. Results (mean ± SD) are derived from at least two separate experiments, counting at least two representative fields in each experiment, with at least 50 cells/field. (C) MDA-MB-435 cells were suspended at 37°C for 30 min, and then either retained in suspension or plated on vitronectin (Vn), laminin-1, or laminin-5 for 45 min. Some samples (lanes 8–10) were preincubated with 1.0 μg/ml PP2 in DMSO for 20 min before plating. Cells were collected and lysed in RIPA, and p130Cas was immunoprecipitated. After SDS-PAGE, samples were blotted with antiphosphotyrosine mAb 4G10. To assess protein loading, the blot was stripped and reblotted with p130Cas-specific antibody. Similar results were seen in multiple experiments.

Mentions: We observed comparable hemidesmosome-like staining for GFP-tagged wild-type and mutant β4 in A431 cells (unpublished data). Because a high level of endogenous β4 precluded further functional studies in A431 cells, we switched to MDA-MB-435 cells, with minimal endogenous β4, for studies of stably expressed wild-type and 7C/S β4. On laminin-5, spreading of 7C/S cells was markedly impaired compared with that of cells with control vector or wild-type β4 (Fig. 5 A, top). All cells spread equally well on vitronectin (Fig. 5 A, bottom). Quantitation of multiple experiments confirmed deficient 7C/S β4 cell spreading on laminin-5 but not vitronectin substrate (Fig. 5 B). A marked defect in tyrosine phosphorylation of p130Cas was also observed for 7C/S β4 cells (Fig. 5 C, lanes 7 and 12) compared with wild-type β4 cells (Fig. 5 C, lanes 6 and 11), when plated on laminin-1 or laminin-5. No defect was seen on control ligand (vitronectin; Fig. 5 C, lanes 3 and 4), and minimal p130Cas phosphorylation was seen for cells in suspension (Fig. 5 C, lanes 1 and 2). In contrast to results with p130Cas, mutant and wild-type β4 showed little difference in phosphorylation of FAK (unpublished data). In concert with cell spreading, p130Cas is typically phosphorylated by a mechanism dependent on Src family kinases (O'Neill et al., 2000). Consistent with this, the Src family kinase inhibitor PP2 (Hanke et al., 1996) abolished both p130Cas phosphorylation (see Fig. 7 A, lanes 8–10) and wild-type β4–MDA-MB-435 cell spreading on laminin-5 (not depicted). Similar spreading and signaling defects were also seen for 7C/S β4–transfected SK-MEL-5 melanoma cells (unpublished data).


Palmitoylation supports assembly and function of integrin-tetraspanin complexes.

Yang X, Kovalenko OV, Tang W, Claas C, Stipp CS, Hemler ME - J. Cell Biol. (2004)

β4 integrin–mediated cell spreading. (A) MDA-MB-435 cells stably expressing vector or wild-type (WT) or 7C/S β4 were held in suspension at 37°C for 30 min, and then were plated on wells coated in PBS at 4°C overnight with either laminin-5 (LN; subpanels A–C) or vitronectin (VN; subpanels D–E), and photographed after 45 min. Cell spreading was monitored using a microscope (Axiovert 135; Carl Zeiss MicroImaging, Inc.) as described previously (Stipp and Hemler, 2000). Bar, 50 μm. (B) Cell spreading was estimated by determining the percentage of cells that were no longer round and phase-bright. The accuracy of this method was validated by quantitation of cell area using Scion Image software, which confirmed that we could readily distinguish cells in which the area had increased by ≥1.2-fold. Results (mean ± SD) are derived from at least two separate experiments, counting at least two representative fields in each experiment, with at least 50 cells/field. (C) MDA-MB-435 cells were suspended at 37°C for 30 min, and then either retained in suspension or plated on vitronectin (Vn), laminin-1, or laminin-5 for 45 min. Some samples (lanes 8–10) were preincubated with 1.0 μg/ml PP2 in DMSO for 20 min before plating. Cells were collected and lysed in RIPA, and p130Cas was immunoprecipitated. After SDS-PAGE, samples were blotted with antiphosphotyrosine mAb 4G10. To assess protein loading, the blot was stripped and reblotted with p130Cas-specific antibody. Similar results were seen in multiple experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172609&req=5

fig5: β4 integrin–mediated cell spreading. (A) MDA-MB-435 cells stably expressing vector or wild-type (WT) or 7C/S β4 were held in suspension at 37°C for 30 min, and then were plated on wells coated in PBS at 4°C overnight with either laminin-5 (LN; subpanels A–C) or vitronectin (VN; subpanels D–E), and photographed after 45 min. Cell spreading was monitored using a microscope (Axiovert 135; Carl Zeiss MicroImaging, Inc.) as described previously (Stipp and Hemler, 2000). Bar, 50 μm. (B) Cell spreading was estimated by determining the percentage of cells that were no longer round and phase-bright. The accuracy of this method was validated by quantitation of cell area using Scion Image software, which confirmed that we could readily distinguish cells in which the area had increased by ≥1.2-fold. Results (mean ± SD) are derived from at least two separate experiments, counting at least two representative fields in each experiment, with at least 50 cells/field. (C) MDA-MB-435 cells were suspended at 37°C for 30 min, and then either retained in suspension or plated on vitronectin (Vn), laminin-1, or laminin-5 for 45 min. Some samples (lanes 8–10) were preincubated with 1.0 μg/ml PP2 in DMSO for 20 min before plating. Cells were collected and lysed in RIPA, and p130Cas was immunoprecipitated. After SDS-PAGE, samples were blotted with antiphosphotyrosine mAb 4G10. To assess protein loading, the blot was stripped and reblotted with p130Cas-specific antibody. Similar results were seen in multiple experiments.
Mentions: We observed comparable hemidesmosome-like staining for GFP-tagged wild-type and mutant β4 in A431 cells (unpublished data). Because a high level of endogenous β4 precluded further functional studies in A431 cells, we switched to MDA-MB-435 cells, with minimal endogenous β4, for studies of stably expressed wild-type and 7C/S β4. On laminin-5, spreading of 7C/S cells was markedly impaired compared with that of cells with control vector or wild-type β4 (Fig. 5 A, top). All cells spread equally well on vitronectin (Fig. 5 A, bottom). Quantitation of multiple experiments confirmed deficient 7C/S β4 cell spreading on laminin-5 but not vitronectin substrate (Fig. 5 B). A marked defect in tyrosine phosphorylation of p130Cas was also observed for 7C/S β4 cells (Fig. 5 C, lanes 7 and 12) compared with wild-type β4 cells (Fig. 5 C, lanes 6 and 11), when plated on laminin-1 or laminin-5. No defect was seen on control ligand (vitronectin; Fig. 5 C, lanes 3 and 4), and minimal p130Cas phosphorylation was seen for cells in suspension (Fig. 5 C, lanes 1 and 2). In contrast to results with p130Cas, mutant and wild-type β4 showed little difference in phosphorylation of FAK (unpublished data). In concert with cell spreading, p130Cas is typically phosphorylated by a mechanism dependent on Src family kinases (O'Neill et al., 2000). Consistent with this, the Src family kinase inhibitor PP2 (Hanke et al., 1996) abolished both p130Cas phosphorylation (see Fig. 7 A, lanes 8–10) and wild-type β4–MDA-MB-435 cell spreading on laminin-5 (not depicted). Similar spreading and signaling defects were also seen for 7C/S β4–transfected SK-MEL-5 melanoma cells (unpublished data).

Bottom Line: There is also a functional connection between CD9 and beta4 integrins, as evidenced by anti-CD9 antibody effects on beta4-dependent cell spreading.Notably, beta4 palmitoylation neither increased localization into "light membrane" fractions of sucrose gradients nor decreased solubility in nonionic detergents-hence it does not promote lipid raft association.Instead, palmitoylation of beta4 (and of the closely associated tetraspanin CD151) promotes CD151-alpha6beta4 incorporation into a network of secondary tetraspanin interactions (with CD9, CD81, CD63, etc.), which provides a novel framework for functional regulation.

View Article: PubMed Central - PubMed

Affiliation: Dana-Farber Cancer Institute and Department of Pathology, Harvard Medical School, Boston, MA 02115, USA.

ABSTRACT
As observed previously, tetraspanin palmitoylation promotes tetraspanin microdomain assembly. Here, we show that palmitoylated integrins (alpha3, alpha6, and beta4 subunits) and tetraspanins (CD9, CD81, and CD63) coexist in substantially overlapping complexes. Removal of beta4 palmitoylation sites markedly impaired cell spreading and signaling through p130Cas on laminin substrate. Also in palmitoylation-deficient beta4, secondary associations with tetraspanins (CD9, CD81, and CD63) were diminished and cell surface CD9 clustering was decreased, whereas core alpha6beta4-CD151 complex formation was unaltered. There is also a functional connection between CD9 and beta4 integrins, as evidenced by anti-CD9 antibody effects on beta4-dependent cell spreading. Notably, beta4 palmitoylation neither increased localization into "light membrane" fractions of sucrose gradients nor decreased solubility in nonionic detergents-hence it does not promote lipid raft association. Instead, palmitoylation of beta4 (and of the closely associated tetraspanin CD151) promotes CD151-alpha6beta4 incorporation into a network of secondary tetraspanin interactions (with CD9, CD81, CD63, etc.), which provides a novel framework for functional regulation.

Show MeSH
Related in: MedlinePlus