Phosphoinositide Binding Inhibits α-Actinin Bundling Activity

Abstract

α-Actinin is an abundant actin-bundling and adhesion protein that directly links actin filaments to integrin receptors. Previously, in platelet-derived growth factor-treated fibroblasts, we demonstrated that phosphoinositides bind to α-actinin, regulating its localization (Greenwood, J. A., Theibert, A. B., Prestwich, G. D., and Murphy-Ullrich, J. E. (2000) J. Cell Biol. 150, 627– 642). In this study, phosphoinositide binding and regulation of α-actinin function is further characterized. Phosphoinositide binding specificity, determined using a protein-lipid overlay procedure, suggests that α-actinin interacts with phosphates on the 4th and 5th position of the inositol head group. Binding assays and mutational analyses demonstrate that phosphoinositides bind to the calponin homology domain 2 of α-actinin. Phosphoinositide binding inhibited the bundling activity of α-actinin by blocking the interaction of the actin-binding domain with actin filaments. Consistent with these results, excessive bundling of actin filaments in an α-actinin with phosphoinositide that the interaction of α-actinin with phosphoinositides actin in the by the to α-Actinin is an abundant actin-bundling and adhesion protein that directly links actin filaments to integrin receptors. Previously, in platelet-derived growth factor-treated fibroblasts, we demonstrated that phosphoinositides bind to α-actinin, regulating its localization (Greenwood, J. A., Theibert, A. B., Prestwich, G. D., and Murphy-Ullrich, J. E. (2000) J. Cell Biol. 150, 627– 642). In this study, phosphoinositide binding and regulation of α-actinin function is further characterized. Phosphoinositide binding specificity, determined using a protein-lipid overlay procedure, suggests that α-actinin interacts with phosphates on the 4th and 5th position of the inositol head group. Binding assays and mutational analyses demonstrate that phosphoinositides bind to the calponin homology domain 2 of α-actinin. Phosphoinositide binding inhibited the bundling activity of α-actinin by blocking the interaction of the actin-binding domain with actin filaments. Consistent with these results, excessive bundling of actin filaments in an α-actinin with phosphoinositide that the interaction of α-actinin with phosphoinositides actin in the by the to interaction of with is regulation of and Cell Biol. In the the and the is a adhesion Cell Biol. of an of that the actin to the integrin adhesion Cell Biol. α-Actinin is an abundant actin-bundling and adhesion protein that a actin and integrin A. J. Cell J. Cell Biol. is that α-actinin function is adhesion and A. A. A. J. Cell is α-actinin is in the an with of the the actin-binding the and the domain A. J. Cell the of α-actinin and the of the that the α-actinin in J. J. J. Biol. α-Actinin to a of and in the actin-binding domain of calponin homology calponin platelet-derived growth phosphoinositide calponin platelet-derived growth phosphoinositide domain this of is binding to actin filaments and bind the domain binding bind J. J. on that the actin-binding of this of that in with actin and 2 the and is the domain J. J. of α-actinin in adhesion is by its localization α-actinin is in its in actin filaments to the domain of E. Cell Biol. of in a adhesion to demonstrate that of the α-actinin actin filaments and in the of the a actin α-actinin is in a that α-actinin is the that with filaments E. G. G. A. J. J. Cell Biol. is that these filaments the of J. Cell Biol. is α-actinin the of these to the of and Cell Biol. and the in G. G. J. to the activity of a of adhesion in A. J. Biol. Cell Biol. J. Biol. A. A. J. Biol. and α-actinin the in regulating the function of these in to using J. Biol. demonstrated that α-actinin and the in α-actinin to J. Biol. to actin-binding binding of to α-actinin to the activity of α-actinin an in that to α-actinin in fibroblasts, with the localization of α-actinin to and actin J. Cell Biol. of and of phosphoinositides adhesion and the actin J. Previously, we demonstrated that platelet-derived growth of the of adhesion and of the actin J. Cell Biol. of by the of α-actinin and integrin and to to bind to α-actinin in with and of the actin the binding of the interaction of α-actinin with the of the integrin on the we that binding to α-actinin in the of and binding in the of these In to the of these we the regulation of α-actinin by and and in the demonstrate that and the bundling activity of α-actinin regulating in the and and J. Biol. and A. J. domain by the of α-actinin by using the Cell Biol. the the and of the in using J. Biol. α-actinin the of by in and using by the Binding binding to α-actinin J. J. J. with 2 of in and the with in with α-actinin in blocking and with with the and with using a the with α-actinin Binding by using and and the overlay In these and by of the phosphoinositides in to to with using an with protein of in with a of and the with a of of in a of of phosphoinositide in to a of and of the bundling of determined by assays J. Cell Biol. a with α-actinin to the and and and the by by and by Binding binding of to using the J. Cell Biol. a with to the and and and the to and actin by and by and and J. Cell Biol. with and using the 2 and J. Cell Biol. using a with a by and protein the and by and with the of to the on α-actinin, and binding using of α-actinin using a binding we that to In to the of α-actinin and the binding in the of of phosphoinositides and inhibited the binding of to in a with an of determined and binding to α-actinin is to that of the homology 2 domain of a phosphoinositide binding J. Biol. and bind to the actin-binding domain of α-actinin with with 2 of and with α-actinin and binding using of binding of to with in the of of we that α-actinin interacts with and in the J. Cell Biol. binding to further of the of the interaction the protein-lipid overlay to an of to a α-Actinin with the in the of binding and bind to the domain of with of the with of Binding using the binding of and to and using of in the and of and the of to the interaction of phosphoinositides with this the a protein and binding in is to the using we and with a protein the of α-actinin, and with and with of the the in of α-actinin binding to of the phosphoinositides to of and and with α-actinin. binding of α-actinin to and overlay assays the binding of and α-actinin to and Binding to and inhibited by of and the the the binding to with the binding we that phosphoinositide binding the of this in a in with a of to of the in the of that and bind to the domain of α-actinin. in the of the phosphoinositides to suggests that and the of the by the Binding of α-Actinin to the of phosphoinositides on α-actinin bundling we a J. Cell Biol. in the of phosphoinositides that the of actin with the of α-actinin, of the actin filaments α-actinin, this the assays to to and of bundling activity by the this and inhibited α-actinin bundling activity in a with In the of α-actinin bundling activity inhibited by and of bundling to a on actin we assays J. Cell Biol. using in and inhibited the binding of the protein to actin filaments. to that the of with we that the of and the binding of to the inositol head of the and on binding to and α-actinin bundling α-actinin with the of α-Actinin to a of actin filaments and the and the by and by the of actin in the using and α-actinin binding to with the of to a of actin filaments and the and the by and by the of in the using Binding to α-Actinin the of phosphoinositide binding on α-actinin function in the and α-actinin in of the in of the Cell α-actinin, to with a actin and in actin the of the actin and of actin filaments in the of and with demonstrated that the and with and on these and the of the bundling assays that with a phosphoinositides α-actinin excessive bundling the the actin-binding that the in α-actinin the in bundling activity in the the in bundling activity of and of actin in the that the directly α-actinin bundling binding to α-actinin actin and by with to of that binding of phosphoinositides to α-actinin actin to a of actin filaments and the and the by and by of the of phosphoinositide binding in bundling the α-actinin to to the in and and in the of the in the In the that and in the the excessive bundling in the to an of a in of the the of actin in a in the this the the using we of of the and in the to that of the in a in actin in the these the that phosphoinositide binding α-actinin bundling of phosphoinositide binding to α-actinin and and the and the by and by with of demonstrated that α-actinin to J. Biol. J. Biol. G. G. G. A. these the the of α-actinin interaction with the of the the regulation of α-actinin function by phosphoinositide and the of phosphoinositide binding to α-actinin on the actin and In this study, we the of α-actinin binding to using a protein-lipid overlay α-Actinin to bind to and with and a binding phosphoinositides α-actinin to and with the of α-actinin with and demonstrated in the suggests that further to the of these in the J. in the regulation of α-actinin. we binding to α-actinin in J. Cell Biol. the and the of the α-Actinin to phosphoinositides on the 4th and 5th of the inositol head that the protein interacts with these In the of and on the position binding to that this is interaction with protein and to demonstrate that phosphoinositides bind to the domain of α-actinin with of J. Biol. the binding domain of α-actinin to of the the inhibited phosphoinositide binding to α-actinin by directly the the inhibited of the phosphoinositides to the binding and assays to the of phosphoinositide binding on α-actinin that and inhibited α-actinin bundling activity by blocking interaction of the actin-binding domain with actin filaments and to that J. Biol. and this with the of J. Biol. that the activity of α-actinin. assays to α-actinin bundling we the the the that the to bundling activity with the of phosphates on the inositol head the and to in binding we that the the on regulating α-actinin bundling to the binding of phosphoinositides α-actinin an α-actinin with phosphoinositide to the on the actin and demonstrated that of phosphoinositide binding to α-actinin in excessive bundling of actin filaments in the and that phosphoinositide binding to α-actinin actin by the of in the localization of and to that phosphoinositides α-actinin the of to this study, we and that binding to α-actinin in and regulating its localization and function and In the that the of α-actinin with is to and binding to α-actinin binding to actin filaments and to localization to that by binding to actin filaments and α-actinin to the the of α-actinin in the regulating of the actin fibroblasts, we demonstrated J. Cell Biol. that binding to α-actinin interaction with the integrin with the of α-actinin In the study, we that binding the interaction of α-actinin with actin filaments bundling activity and we that the binding of to α-actinin an in of the actin by the and α-actinin the actin bundling activity of to a the regulation of by phosphoinositides and and α-actinin binding to actin filaments the actin on the in this and the J. Cell Biol. and to binding to and regulating of α-actinin. that α-actinin is with in the in and binding to α-actinin in and in to the regulation of α-actinin by phosphoinositides the α-actinin with phosphoinositides in this in this interaction of with is regulation of and Cell Biol. In the the and the is a adhesion Cell Biol. of an of that the actin to the integrin adhesion Cell Biol. α-Actinin is an abundant actin-bundling and adhesion protein that a actin and integrin A. J. Cell J. Cell Biol. is that α-actinin function is adhesion and A. A. A. J. Cell is α-actinin is in the α-Actinin an with of the the actin-binding the and the domain A. J. Cell the of α-actinin and the of the that the α-actinin in J. J. J. Biol. α-Actinin to a of and in the actin-binding domain of calponin homology calponin platelet-derived growth phosphoinositide calponin platelet-derived growth phosphoinositide domain this of is binding to actin filaments and bind the domain binding bind J. J. on that the actin-binding of this of that in with actin and 2 the and is the domain J. J. of α-actinin in adhesion is by its localization α-actinin is in its in actin filaments to the domain of E. Cell Biol. of in a adhesion to demonstrate that of the α-actinin actin filaments and in the of the a actin α-actinin is in a that α-actinin is the that with filaments E. G. G. A. J. J. Cell Biol. is that these filaments the of J. Cell Biol. is α-actinin the of these to the of and Cell Biol. and the in G. G. J. to the activity of a of adhesion in A. J. Biol. Cell Biol. J. Biol. A. A. J. Biol. and α-actinin the in regulating the function of these in to using J. Biol. demonstrated that α-actinin and the in α-actinin to J. Biol. to actin-binding binding of to α-actinin to the activity of α-actinin an in that to α-actinin in fibroblasts, with the localization of α-actinin to and actin J. Cell Biol. of and of phosphoinositides adhesion and the actin J. Previously, we demonstrated that platelet-derived growth of the of adhesion and of the actin J. Cell Biol. of by the of α-actinin and integrin and to to bind to α-actinin in with and of the actin the binding of the interaction of α-actinin with the of the integrin on the we that binding to α-actinin in the of and binding in the of these In to the of these we the regulation of α-actinin by and and in the demonstrate that and the bundling activity of α-actinin regulating in the and and J. Biol. and A. J. domain by the of α-actinin by using the Cell Biol. the the and of the in using J. Biol. α-actinin the of by in and using by the Binding binding to α-actinin J. J. J. with 2 of in and the with in with α-actinin in blocking and with with the and with using a the with α-actinin Binding by using and and the overlay In these and by of the phosphoinositides in to to with using an with protein of in with a of and the with a of of in a of of phosphoinositide in to a of and of the bundling of determined by assays J. Cell Biol. a with α-actinin to the and and and the by by and by Binding binding of to using the J. Cell Biol. a with to the and and and the to and actin by and by and and J. Cell Biol. with and using the 2 and J. Cell Biol. using a with a by and protein the and by and with and and J. Biol. and A. J. domain by the of α-actinin by using the Cell Biol. the the and of the in using J. Biol. α-actinin the of by in and using by the Binding binding to α-actinin J. J. J. with 2 of in and the with in with α-actinin in blocking and with with the and with using a the with α-actinin Binding by using and and the overlay In these and by of the phosphoinositides in to to with α-actinin. using an with protein of in with a of and the with a of of in a of of phosphoinositide in to a of and of the bundling of determined by assays J. Cell Biol. a with α-actinin to the and and and the by by and by Binding binding of to using the J. Cell Biol. a with to the and and and the to and actin by and by Cell and and J. Cell Biol. with and using the 2 and J. Cell Biol. using a with a by and protein the and by and with the of to the on α-actinin, and binding using of α-actinin using a binding we that to In to the of α-actinin and the binding in the of of phosphoinositides and inhibited the binding of to in a with an of determined and binding to α-actinin is to that of the homology 2 domain of a phosphoinositide binding J. Biol. we that α-actinin interacts with and in the J. Cell Biol. binding to further of the of the interaction the protein-lipid overlay to an of to a α-Actinin with the in the of binding and bind to the domain of with of the with of Binding using the binding of and to and using of in the and of and the of to the interaction of phosphoinositides with this the a protein and binding in is to the using we and with a protein the of α-actinin, and with and with of the the in of α-actinin binding to of the phosphoinositides to of and and with α-actinin. binding of α-actinin to and overlay assays the binding of and α-actinin to and Binding to and inhibited by of and the the the binding to with the binding we that phosphoinositide binding the of this in a in with a of to of the in the of that and bind to the domain of α-actinin. in the of the phosphoinositides to suggests that and the of the by the Binding of α-Actinin to the of phosphoinositides on α-actinin bundling we a J. Cell Biol. in the of phosphoinositides that the of actin with the of α-actinin, of the actin filaments α-actinin, this the assays to to and of bundling activity by the this and inhibited α-actinin bundling activity in a with In the of α-actinin bundling activity inhibited by and of bundling to a on actin we assays J. Cell Biol. using in and inhibited the binding of the protein to actin filaments. to that the of with we that the of and the binding of to the inositol head of the and on binding to and α-actinin bundling α-actinin with the of α-Actinin to a of actin filaments and the and the by and by the of actin in the using and α-actinin binding to with the of to a of actin filaments and the and the by and by the of in the using Binding to α-Actinin the of phosphoinositide binding on α-actinin function in the and α-actinin in of the in of the Cell α-actinin, to with a actin and in actin the of the actin and of actin filaments in the of and with demonstrated that the and with and on these and the of the bundling assays that with a phosphoinositides α-actinin excessive bundling the the actin-binding that the in α-actinin the in bundling activity in the the in bundling activity of and of actin in the that the directly α-actinin bundling binding to α-actinin actin and by with to of that binding of phosphoinositides to α-actinin actin to a of actin filaments and the and the by and by of the of phosphoinositide binding in bundling the α-actinin to to the in and and in the of the in the In the that and in the the excessive bundling in the to an of a in of the the of actin in a in the this the the using we of of the and in the to that of the in a in actin in the these the that phosphoinositide binding α-actinin bundling of phosphoinositide binding to α-actinin and and the and the by and by with of the of to the on α-actinin, and binding using of α-actinin using a binding we that to In to the of α-actinin and the binding in the of of phosphoinositides and inhibited the binding of to in a with an of determined and binding to α-actinin is to that of the homology 2 domain of a phosphoinositide binding J. Biol. we that α-actinin interacts with and in the J. Cell Biol. binding to further of the of the interaction the protein-lipid overlay to an of to a α-Actinin with the in the of binding and In to the interaction of phosphoinositides with this the a protein and binding in is to the using we and with a protein the of α-actinin, and with and with of the the in of α-actinin binding to of the phosphoinositides to of and and with α-actinin. binding of α-actinin to and overlay assays the binding of and α-actinin to and Binding to and inhibited by of and the the the binding to with the binding we that phosphoinositide binding the of this in a in with a of to of the in the of that and bind to the domain of α-actinin. in the of the phosphoinositides to suggests that and the of the by the Binding of α-Actinin to the of phosphoinositides on α-actinin bundling we a J. Cell Biol. in the of phosphoinositides that the of actin with the of α-actinin, of the actin filaments α-actinin, this the assays to to and of bundling activity by the this and inhibited α-actinin bundling activity in a with In the of α-actinin bundling activity inhibited by and of bundling to a on actin we assays J. Cell Biol. using in and inhibited the binding of the protein to actin filaments. to that the of with we that the of and the binding of to the inositol head of the and on binding to Phosphoinositide Binding to α-Actinin the of phosphoinositide binding on α-actinin function in the and α-actinin in of the in of the Cell α-actinin, to with a actin and in actin the of the actin and of actin filaments in the of and with demonstrated that the and with and on these and the of the bundling assays that with a phosphoinositides α-actinin excessive bundling the the actin-binding that the in α-actinin the in bundling activity in the the in bundling activity of and of actin in the that the directly α-actinin bundling the of phosphoinositide binding in bundling the α-actinin to to the in and and in the of the in the In the that and in the the excessive bundling in the to an of a in of the the of actin in a in the this the the using we of of the and in the to that of the in a in actin in the these the that phosphoinositide binding α-actinin bundling demonstrated that α-actinin to J. Biol. J. Biol. G. G. G. A. these the the of α-actinin interaction with the of the the regulation of α-actinin function by phosphoinositide and the of phosphoinositide binding to α-actinin on the actin and In this study, we the of α-actinin binding to using a protein-lipid overlay α-Actinin to bind to and with and a binding phosphoinositides α-actinin to and with the of α-actinin with and demonstrated in the suggests that further to the of these in the J. in the regulation of α-actinin. we binding to α-actinin in J. Cell Biol. the and the of the α-Actinin to phosphoinositides on the 4th and 5th of the inositol head that the protein interacts with these In the of and on the position binding to that this is interaction with protein and to demonstrate that phosphoinositides bind to the domain of α-actinin with of J. Biol. the binding domain of α-actinin to of the the inhibited phosphoinositide binding to α-actinin by directly the the inhibited of the phosphoinositides to the binding and assays to the of phosphoinositide binding on α-actinin that and inhibited α-actinin bundling activity by blocking interaction of the actin-binding domain with actin filaments and to that J. Biol. and this with the of J. Biol. that the activity of α-actinin. assays to α-actinin bundling we the the the that the to bundling activity with the of phosphates on the inositol head the and to in binding we that the the on regulating α-actinin bundling to the binding of phosphoinositides α-actinin an α-actinin with phosphoinositide to the on the actin and demonstrated that of phosphoinositide binding to α-actinin in excessive bundling of actin filaments in the and that phosphoinositide binding to α-actinin actin by the of in the localization of and to that phosphoinositides α-actinin the of to this study, we and that binding to α-actinin in and regulating its localization and function and In the that the of α-actinin with is to and binding to α-actinin binding to actin filaments and to localization to that by binding to actin filaments and α-actinin to the the of α-actinin in the regulating of the actin fibroblasts, we demonstrated J. Cell Biol. that binding to α-actinin interaction with the integrin with the of α-actinin In the study, we that binding the interaction of α-actinin with actin filaments bundling activity and we that the binding of to α-actinin an in of the actin by the and α-actinin the actin bundling activity of to a the regulation of by phosphoinositides and and α-actinin binding to actin filaments the actin on the in this and the J. Cell Biol. and to binding to and regulating of α-actinin. that α-actinin is with in the in and binding to α-actinin in and in to the regulation of α-actinin by phosphoinositides the α-actinin with phosphoinositides in this in this demonstrated that α-actinin to J. Biol. J. Biol. G. G. G. A. these the the of α-actinin interaction with the of the the regulation of α-actinin function by phosphoinositide and the of phosphoinositide binding to α-actinin on the actin and In this study, we the of α-actinin binding to using a protein-lipid overlay α-Actinin to bind to and with and a binding phosphoinositides α-actinin to and with the of α-actinin with and demonstrated in the suggests that further to the of these in the J. in the regulation of α-actinin. we binding to α-actinin in J. Cell Biol. the and the of the α-Actinin to phosphoinositides on the 4th and 5th of the inositol head that the protein interacts with these In the of and on the position binding to that this is interaction with α-actinin. protein and to demonstrate that phosphoinositides bind to the domain of α-actinin with of J. Biol. the binding domain of α-actinin to of the the inhibited phosphoinositide binding to α-actinin by directly the the inhibited of the phosphoinositides to the binding and assays to the of phosphoinositide binding on α-actinin that and inhibited α-actinin bundling activity by blocking interaction of the actin-binding domain with actin filaments and to that J. Biol. and this with the of J. Biol. that the activity of α-actinin. assays to α-actinin bundling we the the the that the to bundling activity with the of phosphates on the inositol head the and to in binding we that the the on regulating α-actinin bundling to the binding of phosphoinositides α-actinin an α-actinin with phosphoinositide to the on the actin and demonstrated that of phosphoinositide binding to α-actinin in excessive bundling of actin filaments in the and that phosphoinositide binding to α-actinin actin by the of in the localization of and to that phosphoinositides α-actinin the of to this study, we and that binding to α-actinin in and regulating its localization and function and In the that the of α-actinin with is to and binding to α-actinin binding to actin filaments and to localization to that by binding to actin filaments and α-actinin to the the of α-actinin in the regulating of the actin In fibroblasts, we demonstrated J. Cell Biol. that binding to α-actinin interaction with the integrin with the of α-actinin In the study, we that binding the interaction of α-actinin with actin filaments bundling activity and we that the binding of to α-actinin an in of the actin by the and α-actinin the actin bundling activity of α-actinin. to a the regulation of by phosphoinositides and and α-actinin binding to actin filaments the actin on the in this and the J. Cell Biol. and to binding to and regulating of α-actinin. that α-actinin is with in the in and binding to α-actinin in and in to the regulation of α-actinin by phosphoinositides the α-actinin with phosphoinositides in this in this of the and A. and of A. in by the and and Cell of the and by the of