Limits...
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.

Show MeSH

Related in: MedlinePlus

ICAP-1 inhibits and binds Cdc42. (A) COS cells transiently transfected with myc-Cdc42 together 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). Cdc42 activity assay was performed as described in Materials and methods using a GST-PAK-CD fusion protein that selectively binds to GTP-Cdc42. The bound GTP-Cdc42 was analyzed by Western blotting with a polyclonal antibody to Cdc42. To probe for Cdc42 and ICAP-1 expression, total cell lysates were blotted with the corresponding antibodies. (B) The amount of GTP-bound and total Cdc42 (A, bottom band of the doublets) was quantified by densitometric analysis, and the activation level of Cdc42 was expressed as a ratio between the values of GTP-bound and total Cdc42. Comparable results were obtained in three independent experiments. (C) Total protein extract of COS cells transfected with GST-Cdc42 was loaded on Sepharose coupled with MBP–ICAP-1 fusion protein or MBP as control. The proteins eluted with glycine-HCl, pH 3.0, buffer (fractions numbers 1-2-3-4) were analyzed by Western blotting with polyclonal antibody against Cdc42. Note that in A, Cdc42 is detected as a doublet of bands; comparison of the electrophoretic mobility indicates that the bottom band of the doublet in A comigrates with the band in C. The top band of the doublet is likely to represent an incompletely processed form of the GTPase. (D) Western blotting with an antibody against Cdc42 of the pooled fractions eluted from MBP–ICAP-1-Sepharose columns (right). The columns were loaded with equal amounts of GST-Cdc42 fusion protein obtained from either the membrane fraction of pCEFL-GST-Cdc42–transfected COS cells (a), the cytoplasmic fraction of the same cells (b), or bacterial lysate of E. coli producing the GST-Cdc42 fusion protein (c). The amount of fusion proteins loaded on the column are shown on the left as control for equal loading.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2199222&req=5

fig3: ICAP-1 inhibits and binds Cdc42. (A) COS cells transiently transfected with myc-Cdc42 together 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). Cdc42 activity assay was performed as described in Materials and methods using a GST-PAK-CD fusion protein that selectively binds to GTP-Cdc42. The bound GTP-Cdc42 was analyzed by Western blotting with a polyclonal antibody to Cdc42. To probe for Cdc42 and ICAP-1 expression, total cell lysates were blotted with the corresponding antibodies. (B) The amount of GTP-bound and total Cdc42 (A, bottom band of the doublets) was quantified by densitometric analysis, and the activation level of Cdc42 was expressed as a ratio between the values of GTP-bound and total Cdc42. Comparable results were obtained in three independent experiments. (C) Total protein extract of COS cells transfected with GST-Cdc42 was loaded on Sepharose coupled with MBP–ICAP-1 fusion protein or MBP as control. The proteins eluted with glycine-HCl, pH 3.0, buffer (fractions numbers 1-2-3-4) were analyzed by Western blotting with polyclonal antibody against Cdc42. Note that in A, Cdc42 is detected as a doublet of bands; comparison of the electrophoretic mobility indicates that the bottom band of the doublet in A comigrates with the band in C. The top band of the doublet is likely to represent an incompletely processed form of the GTPase. (D) Western blotting with an antibody against Cdc42 of the pooled fractions eluted from MBP–ICAP-1-Sepharose columns (right). The columns were loaded with equal amounts of GST-Cdc42 fusion protein obtained from either the membrane fraction of pCEFL-GST-Cdc42–transfected COS cells (a), the cytoplasmic fraction of the same cells (b), or bacterial lysate of E. coli producing the GST-Cdc42 fusion protein (c). The amount of fusion proteins loaded on the column are shown on the left as control for equal loading.

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 Cdc42. (A) COS cells transiently transfected with myc-Cdc42 together 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). Cdc42 activity assay was performed as described in Materials and methods using a GST-PAK-CD fusion protein that selectively binds to GTP-Cdc42. The bound GTP-Cdc42 was analyzed by Western blotting with a polyclonal antibody to Cdc42. To probe for Cdc42 and ICAP-1 expression, total cell lysates were blotted with the corresponding antibodies. (B) The amount of GTP-bound and total Cdc42 (A, bottom band of the doublets) was quantified by densitometric analysis, and the activation level of Cdc42 was expressed as a ratio between the values of GTP-bound and total Cdc42. Comparable results were obtained in three independent experiments. (C) Total protein extract of COS cells transfected with GST-Cdc42 was loaded on Sepharose coupled with MBP–ICAP-1 fusion protein or MBP as control. The proteins eluted with glycine-HCl, pH 3.0, buffer (fractions numbers 1-2-3-4) were analyzed by Western blotting with polyclonal antibody against Cdc42. Note that in A, Cdc42 is detected as a doublet of bands; comparison of the electrophoretic mobility indicates that the bottom band of the doublet in A comigrates with the band in C. The top band of the doublet is likely to represent an incompletely processed form of the GTPase. (D) Western blotting with an antibody against Cdc42 of the pooled fractions eluted from MBP–ICAP-1-Sepharose columns (right). The columns were loaded with equal amounts of GST-Cdc42 fusion protein obtained from either the membrane fraction of pCEFL-GST-Cdc42–transfected COS cells (a), the cytoplasmic fraction of the same cells (b), or bacterial lysate of E. coli producing the GST-Cdc42 fusion protein (c). The amount of fusion proteins loaded on the column are shown on the left as control for equal loading.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199222&req=5

fig3: ICAP-1 inhibits and binds Cdc42. (A) COS cells transiently transfected with myc-Cdc42 together 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). Cdc42 activity assay was performed as described in Materials and methods using a GST-PAK-CD fusion protein that selectively binds to GTP-Cdc42. The bound GTP-Cdc42 was analyzed by Western blotting with a polyclonal antibody to Cdc42. To probe for Cdc42 and ICAP-1 expression, total cell lysates were blotted with the corresponding antibodies. (B) The amount of GTP-bound and total Cdc42 (A, bottom band of the doublets) was quantified by densitometric analysis, and the activation level of Cdc42 was expressed as a ratio between the values of GTP-bound and total Cdc42. Comparable results were obtained in three independent experiments. (C) Total protein extract of COS cells transfected with GST-Cdc42 was loaded on Sepharose coupled with MBP–ICAP-1 fusion protein or MBP as control. The proteins eluted with glycine-HCl, pH 3.0, buffer (fractions numbers 1-2-3-4) were analyzed by Western blotting with polyclonal antibody against Cdc42. Note that in A, Cdc42 is detected as a doublet of bands; comparison of the electrophoretic mobility indicates that the bottom band of the doublet in A comigrates with the band in C. The top band of the doublet is likely to represent an incompletely processed form of the GTPase. (D) Western blotting with an antibody against Cdc42 of the pooled fractions eluted from MBP–ICAP-1-Sepharose columns (right). The columns were loaded with equal amounts of GST-Cdc42 fusion protein obtained from either the membrane fraction of pCEFL-GST-Cdc42–transfected COS cells (a), the cytoplasmic fraction of the same cells (b), or bacterial lysate of E. coli producing the GST-Cdc42 fusion protein (c). The amount of fusion proteins loaded on the column are shown on the left as control for equal loading.
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