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The integrin cytoplasmic domain-associated protein ICAP-1 binds and regulates Rho family GTPases during cell spreading.

Degani S, Balzac F, Brancaccio M, Guazzone S, Retta SF, Silengo L, Eva A, Tarone G - J. Cell Biol. (2002)

Bottom Line: Using two-hybrid screening, we isolated the integrin cytoplasmic domain-associated protein (ICAP-1), an interactor for the COOH terminal region of the beta1A integrin cytoplasmic domain.ICAP-1 expression strongly prevents NIH3T3 cell spreading on extracellular matrix.This inhibition is transient and can be counteracted by coexpression of a constitutively activated mutant of Cdc42, suggesting that ICAP-1 acts upstream of this GTPase.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Biology and Biochemistry, University of Torino, 10126 Torino, Italy.

ABSTRACT
Using two-hybrid screening, we isolated the integrin cytoplasmic domain-associated protein (ICAP-1), an interactor for the COOH terminal region of the beta1A integrin cytoplasmic domain. To investigate the role of ICAP-1 in integrin-mediated adhesive function, we expressed the full-length molecule in NIH3T3 cells. ICAP-1 expression strongly prevents NIH3T3 cell spreading on extracellular matrix. This inhibition is transient and can be counteracted by coexpression of a constitutively activated mutant of Cdc42, suggesting that ICAP-1 acts upstream of this GTPase. In addition, we found that ICAP-1 binds both to Cdc42 and Rac1 in vitro, and its expression markedly inhibits activation of these GTPases during integrin-mediated cell adhesion to fibronectin as detected by PAK binding assay. In the attempt to define the molecular mechanism of this inhibition, we show that ICAP-1 reduces both the intrinsic and the exchange factor-induced dissociation of GDP from Cdc42; moreover, purified ICAP-1 displaces this GTPase from cellular membranes. Together, these data show for the first time that ICAP-1 regulates Rho family GTPases during integrin-mediated cell matrix adhesion, acting as guanine dissociation inhibitor.

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ICAP-1 inhibits and binds Rac1. (A) COS cells transiently transfected with control vector or myc–ICAP-1 were suspended by trypsin treatment, kept in suspension 2 h 30 min in serum-free DME (Su), and plated on fibronectin (5 μg/ml) for 1 h (Ad). Rac1 activation level was evaluated as described in Materials and methods using a GST-PAK-CD fusion protein that selectively binds to GTP-Rac1. The bound GTP-Rac1 was analyzed by Western blotting with a polyclonal antibody to Rac1. To probe for Rac1 and ICAP-1 expression, total cell lysates were blotted with the corresponding antibodies. (B) The amount of GTP-bound and total Rac1 (A) was quantified by densitometric analysis; the activation level of Rac1 is expressed as a ratio between the values of GTP-bound and total Rac1. Comparable results were obtained in three independent experiments. (C) Total lysate of COS cells transfected with myc-Rac1 was loaded on Sepharose coupled with MBP–ICAP-1 fusion protein or MBP as control. The proteins eluted with glycine-HCl, pH 3, buffer (fractions numbers 1-2-3-4) were analyzed by Western blotting with a monoclonal antibody against Rac1.
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fig4: ICAP-1 inhibits and binds Rac1. (A) COS cells transiently transfected with control vector or myc–ICAP-1 were suspended by trypsin treatment, kept in suspension 2 h 30 min in serum-free DME (Su), and plated on fibronectin (5 μg/ml) for 1 h (Ad). Rac1 activation level was evaluated as described in Materials and methods using a GST-PAK-CD fusion protein that selectively binds to GTP-Rac1. The bound GTP-Rac1 was analyzed by Western blotting with a polyclonal antibody to Rac1. To probe for Rac1 and ICAP-1 expression, total cell lysates were blotted with the corresponding antibodies. (B) The amount of GTP-bound and total Rac1 (A) was quantified by densitometric analysis; the activation level of Rac1 is expressed as a ratio between the values of GTP-bound and total Rac1. Comparable results were obtained in three independent experiments. (C) Total lysate of COS cells transfected with myc-Rac1 was loaded on Sepharose coupled with MBP–ICAP-1 fusion protein or MBP as control. The proteins eluted with glycine-HCl, pH 3, buffer (fractions numbers 1-2-3-4) were analyzed by Western blotting with a monoclonal antibody against Rac1.

Mentions: Based on the results described above, we tested the possibility that ICAP-1 could regulate the activity of Rho family GTPases. To measure the activation state of Cdc42 and Rac1, the GTP-bound form of these proteins was isolated using the CRIB domain of the effector protein PAK (Sander et al., 1998). Cells transfected with control vector or ICAP-1 were either maintained in suspension or plated on fibronectin, and cell extracts were processed as described in Materials and methods. COS cells were used in these experiments in order to achieve high level of expression. As shown in Fig. 3 A and Fig. 4 A, the amount of GTP-bound GTPases is increased upon cell adhesion to fibronectin, indicating that both Cdc42 and Rac1 are activated during cell spreading on fibronectin. However, expression of ICAP-1 in these cells strongly reduced the level of GTP-bound form of both GTPases (Fig. 3 and Fig. 4, A and B).


The integrin cytoplasmic domain-associated protein ICAP-1 binds and regulates Rho family GTPases during cell spreading.

Degani S, Balzac F, Brancaccio M, Guazzone S, Retta SF, Silengo L, Eva A, Tarone G - J. Cell Biol. (2002)

ICAP-1 inhibits and binds Rac1. (A) COS cells transiently transfected with control vector or myc–ICAP-1 were suspended by trypsin treatment, kept in suspension 2 h 30 min in serum-free DME (Su), and plated on fibronectin (5 μg/ml) for 1 h (Ad). Rac1 activation level was evaluated as described in Materials and methods using a GST-PAK-CD fusion protein that selectively binds to GTP-Rac1. The bound GTP-Rac1 was analyzed by Western blotting with a polyclonal antibody to Rac1. To probe for Rac1 and ICAP-1 expression, total cell lysates were blotted with the corresponding antibodies. (B) The amount of GTP-bound and total Rac1 (A) was quantified by densitometric analysis; the activation level of Rac1 is expressed as a ratio between the values of GTP-bound and total Rac1. Comparable results were obtained in three independent experiments. (C) Total lysate of COS cells transfected with myc-Rac1 was loaded on Sepharose coupled with MBP–ICAP-1 fusion protein or MBP as control. The proteins eluted with glycine-HCl, pH 3, buffer (fractions numbers 1-2-3-4) were analyzed by Western blotting with a monoclonal antibody against Rac1.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199222&req=5

fig4: ICAP-1 inhibits and binds Rac1. (A) COS cells transiently transfected with control vector or myc–ICAP-1 were suspended by trypsin treatment, kept in suspension 2 h 30 min in serum-free DME (Su), and plated on fibronectin (5 μg/ml) for 1 h (Ad). Rac1 activation level was evaluated as described in Materials and methods using a GST-PAK-CD fusion protein that selectively binds to GTP-Rac1. The bound GTP-Rac1 was analyzed by Western blotting with a polyclonal antibody to Rac1. To probe for Rac1 and ICAP-1 expression, total cell lysates were blotted with the corresponding antibodies. (B) The amount of GTP-bound and total Rac1 (A) was quantified by densitometric analysis; the activation level of Rac1 is expressed as a ratio between the values of GTP-bound and total Rac1. Comparable results were obtained in three independent experiments. (C) Total lysate of COS cells transfected with myc-Rac1 was loaded on Sepharose coupled with MBP–ICAP-1 fusion protein or MBP as control. The proteins eluted with glycine-HCl, pH 3, buffer (fractions numbers 1-2-3-4) were analyzed by Western blotting with a monoclonal antibody against Rac1.
Mentions: Based on the results described above, we tested the possibility that ICAP-1 could regulate the activity of Rho family GTPases. To measure the activation state of Cdc42 and Rac1, the GTP-bound form of these proteins was isolated using the CRIB domain of the effector protein PAK (Sander et al., 1998). Cells transfected with control vector or ICAP-1 were either maintained in suspension or plated on fibronectin, and cell extracts were processed as described in Materials and methods. COS cells were used in these experiments in order to achieve high level of expression. As shown in Fig. 3 A and Fig. 4 A, the amount of GTP-bound GTPases is increased upon cell adhesion to fibronectin, indicating that both Cdc42 and Rac1 are activated during cell spreading on fibronectin. However, expression of ICAP-1 in these cells strongly reduced the level of GTP-bound form of both GTPases (Fig. 3 and Fig. 4, A and B).

Bottom Line: Using two-hybrid screening, we isolated the integrin cytoplasmic domain-associated protein (ICAP-1), an interactor for the COOH terminal region of the beta1A integrin cytoplasmic domain.ICAP-1 expression strongly prevents NIH3T3 cell spreading on extracellular matrix.This inhibition is transient and can be counteracted by coexpression of a constitutively activated mutant of Cdc42, suggesting that ICAP-1 acts upstream of this GTPase.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Biology and Biochemistry, University of Torino, 10126 Torino, Italy.

ABSTRACT
Using two-hybrid screening, we isolated the integrin cytoplasmic domain-associated protein (ICAP-1), an interactor for the COOH terminal region of the beta1A integrin cytoplasmic domain. To investigate the role of ICAP-1 in integrin-mediated adhesive function, we expressed the full-length molecule in NIH3T3 cells. ICAP-1 expression strongly prevents NIH3T3 cell spreading on extracellular matrix. This inhibition is transient and can be counteracted by coexpression of a constitutively activated mutant of Cdc42, suggesting that ICAP-1 acts upstream of this GTPase. In addition, we found that ICAP-1 binds both to Cdc42 and Rac1 in vitro, and its expression markedly inhibits activation of these GTPases during integrin-mediated cell adhesion to fibronectin as detected by PAK binding assay. In the attempt to define the molecular mechanism of this inhibition, we show that ICAP-1 reduces both the intrinsic and the exchange factor-induced dissociation of GDP from Cdc42; moreover, purified ICAP-1 displaces this GTPase from cellular membranes. Together, these data show for the first time that ICAP-1 regulates Rho family GTPases during integrin-mediated cell matrix adhesion, acting as guanine dissociation inhibitor.

Show MeSH
Related in: MedlinePlus