Phosphatidylinositol Phosphate Kinase Type 1γ and β1-Integrin Cytoplasmic Domain Bind to the Same Region in the Talin FERM Domain

Abstract

Talin is an essential component of focal adhesions that couples β-integrin cytodomains to F-actin and provides a scaffold for signaling proteins. Recently, the integrin β3 cytodomain and phosphatidylinositol phosphate (PIP) kinase type 1γ (a phosphatidylinositol 4,5-bisphosphate-synthesizing enzyme) were shown to bind to the talin FERM domain (subdomain F3). We have characterized the PIP kinase-binding site by NMR using a 15N-labeled talin F2F3 polypeptide. A PIP kinase peptide containing the minimal talin-binding site formed a 1:1 complex with F2F3, causing a substantial number of chemical shift changes. In particular, two of the three Arg residues (Arg339 and Arg358), four of eight Ile residues, and one of seven Val residues in F3 were affected. Although a R339A mutation did not affect the exchange kinetics, R358A or R358K mutations markedly weakened binding. The K d for the interaction determined by Trp fluorescence was 6 μm, and the R358A mutation increased the K d to 35 μm. Comparison of these results with those of the crystal structure of a β3-integrin cytodomain talin F2F3 chimera shows that both PIP kinase and integrins bind to the same surface of the talin F3 subdomain. Indeed, binding of talin present in rat brain extracts to a glutathione S-transferase integrin β1-cytodomain polypeptide was inhibited by the PIP kinase peptide. The results suggest that ternary complex formation with a single talin FERM domain is unlikely, although both integrins and PIP kinase may bind simultaneously to the talin anti-parallel dimer. Talin is an essential component of focal adhesions that couples β-integrin cytodomains to F-actin and provides a scaffold for signaling proteins. Recently, the integrin β3 cytodomain and phosphatidylinositol phosphate (PIP) kinase type 1γ (a phosphatidylinositol 4,5-bisphosphate-synthesizing enzyme) were shown to bind to the talin FERM domain (subdomain F3). We have characterized the PIP kinase-binding site by NMR using a 15N-labeled talin F2F3 polypeptide. A PIP kinase peptide containing the minimal talin-binding site formed a 1:1 complex with F2F3, causing a substantial number of chemical shift changes. In particular, two of the three Arg residues (Arg339 and Arg358), four of eight Ile residues, and one of seven Val residues in F3 were affected. Although a R339A mutation did not affect the exchange kinetics, R358A or R358K mutations markedly weakened binding. The K d for the interaction determined by Trp fluorescence was 6 μm, and the R358A mutation increased the K d to 35 μm. Comparison of these results with those of the crystal structure of a β3-integrin cytodomain talin F2F3 chimera shows that both PIP kinase and integrins bind to the same surface of the talin F3 subdomain. Indeed, binding of talin present in rat brain extracts to a glutathione S-transferase integrin β1-cytodomain polypeptide was inhibited by the PIP kinase peptide. The results suggest that ternary complex formation with a single talin FERM domain is unlikely, although both integrins and PIP kinase may bind simultaneously to the talin anti-parallel dimer. Integrins are the principal family of receptors mediating cellular interactions with the extracellular matrix (ECM). 1The abbreviations used are: ECM, extracellular matrix; FA, focal adhesion; PIP, phosphatidylinositol phosphate; PIP2, phosphatidylinositol 4,5-bisphosphate; GST, glutathione S-transferase; HSQC, heteronuclear single-quantum correlation.1The abbreviations used are: ECM, extracellular matrix; FA, focal adhesion; PIP, phosphatidylinositol phosphate; PIP2, phosphatidylinositol 4,5-bisphosphate; GST, glutathione S-transferase; HSQC, heteronuclear single-quantum correlation. As such, they support a wide variety of processes that are dependent on cell-ECM interactions including cell proliferation, the suppression of apoptosis, cell migration, and the organization of cells into tissues (1Hynes R.O. Cell. 2002; 110: 673-687Abstract Full Text Full Text PDF PubMed Scopus (6809) Google Scholar, 2Hemler M.E. Kreis T. Vale R. Guidebook to the Extracellular Matrix, Anchor and Adhesion Proteins. 2nd Ed. Oxford University Press, Oxford, UK1999: 196-212Google Scholar). Integrins are noncovalent αβ-heterodimeric type I transmembrane proteins that are frequently linked to the actomyosin contractile apparatus within the cell via cytoskeletal proteins such as talin (3Calderwood D.A. Yan B. de Pereda J.M. Alvarez B.G. Fujioka Y. Liddington R.C. Ginsberg M.H. J. Biol. Chem. 2002; 277: 21749-21758Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar), filamin (4Calderwood D.A. Huttenlocher A. Kiosses W.B. Rose D.M. Woodside D.G. Schwartz M.A. Ginsberg M.H. Nat. Cell Biol. 2001; 3: 1060-1068Crossref PubMed Scopus (185) Google Scholar), and α-actinin (5Otey C.A. Vasquez G.B. Burridge K. Erickson B.W. J. Biol. Chem. 1993; 268: 21193-21197Abstract Full Text PDF PubMed Google Scholar, 6Rajfur Z. Roy P. Otey C.A. Romer L. Jacobson K. Nat. Cell Biol. 2002; 4: 286-293Crossref PubMed Scopus (167) Google Scholar). Although it is self-evident that such a link is necessary to support integrin-mediated cell migration, these cytoskeletal proteins also recruit, either directly or indirectly, numerous signaling proteins to integrin cytodomains, which regulate cell adhesion, motility, and the signaling pathways that control cell proliferation and apoptosis (7Calderwood D.A. Shattil S.J. Ginsberg M.H. J. Biol. Chem. 2000; 275: 22607-22610Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar, 8Giancotti F.G. Ruoslahti E. Science. 1999; 285: 1028-1032Crossref PubMed Scopus (3794) Google Scholar, 9Schoenwaelder S.M. Burridge K. Curr. Opin. Cell Biol. 1999; 11: 274-286Crossref PubMed Scopus (649) Google Scholar). Moreover, expression of an N-terminal fragment of talin, which binds to the β-integrin cytodomain, results in activation of αIIbβ3-integrin expressed in Chinese hamster ovary cells (3Calderwood D.A. Yan B. de Pereda J.M. Alvarez B.G. Fujioka Y. Liddington R.C. Ginsberg M.H. J. Biol. Chem. 2002; 277: 21749-21758Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar), suggesting that talin directly regulates integrin affinity, possibly by disrupting the interaction between the highly conserved membrane-proximal regions of the αβ-subunit cytodomains (10O'Toole T.E. Katagiri Y. Faull R.J. Peter K. Tamura R. Quaranta V. Loftus J.C. Shattil S.J. Ginsberg M.H. J. Cell Biol. 1994; 124: 1047-1059Crossref PubMed Scopus (578) Google Scholar). Talin co-localizes with integrins in cell-ECM junctions (focal adhesions; FAs), and microinjection of talin antibodies leads to the disruption of these structures and associated actin stress fibers (11Nuckolls G.H. Romer L.H. Burridge K. J. Cell Sci. 1992; 102: 753-762Crossref PubMed Google Scholar, 12Bolton S.J. Barry S.T. Mosley H. Patel B. Jockusch B.M. Wilkinson J.M. Critchley D.R. Cell Motil. Cytoskelet. 1997; 36: 363-376Crossref PubMed Scopus (39) Google Scholar). Down-regulation of talin expression in HeLa cells using antisense RNA slows down the rate of cell spreading and leads to a reduction in the size of FAs (13Albiges-Rizo C. Frachet P. Block M.R. J. Cell Sci. 1995; 108: 3317-3329Crossref PubMed Google Scholar), and mouse embryonic stem cells in which both copies of the talin gene have been disrupted showed spreading defects and were unable to assemble FAs (14Priddle H. Hemmings L. Monkley S. Woods A. Patel B. Sutton D. Dunn G.A. Zicha D. Critchley D.R. J. Cell Biol. 1998; 142: 1121-1133Crossref PubMed Scopus (148) Google Scholar). However, analysis of talin function in mammalian systems is complicated by the recent discovery of a second talin gene (TLN2), which we have shown encodes a closely related protein (also 2541 residues, 74% identity) but with a more restricted pattern of expression (15Monkley S.J. Pritchard C.A. Critchley D.R. Biochem. Biophys. Res. Commun. 2001; 286: 880-885Crossref PubMed Scopus (89) Google Scholar). Nevertheless, mouse embryos with the TLN1 (–/–) genotype failed to complete gastrulation (16Monkley S.J. Zho X.-H. Kinston S.J. Giblett S.M. Hemmings L. Priddle H. Brown J.E. Pritchard C.A. Critchley D.R. Fassler R Dev. Dynamics. 2000; 219: 560-574Crossref PubMed Scopus (173) Google Scholar), probably because of a failure of mesoderm migration, and in Drosophila, deletion of the single talin gene gives rise to phenotypes very similar to integrin null alleles (17Brown N.H. Gregory S.L. Rickoll W.L. Fessler L.I. Prout M. White R.A. Fristrom J.W. Dev. Cell. 2002; 3: 569-579Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar). The known biochemical properties of talin are also consistent with a role in linking integrins to F-actin. It is a large (270 kDa) flexible rod-shaped (600 nm) (18Winkler J. Lunsdorf H. Jockusch B.M. Eur. J. Biochem. 1997; 243: 430-436Crossref PubMed Scopus (45) Google Scholar) actin-cross-linking protein (19Schmidt J.M. Zhang J. Lee H.-S. Stromer M.H. Robson R.M. Arch. Biochem. Biophys. 1999; 366: 139-150Crossref PubMed Scopus (35) Google Scholar) 2541 residues in length (20Rees D.J.G. Ades S.E. Singer S.J. Hynes R.O. Nature. 1990; 347: 685-689Crossref PubMed Scopus (240) Google Scholar, 21Hemmings L. Rees D.J.G. Ohanian V. Bolton S.J. Gilmore A.P. Patel N. Priddle H. Trevithick J.E. Hynes R.O. Critchley D.R. J. Cell Sci. 1996; 109: 2715-2726Crossref PubMed Google Scholar). The globular talin head contains a region (residues 86–410) homologous to the N-terminal FERM domain of the band 4.1, ezrin, radixin, moesin family of cytoskeletal proteins (20Rees D.J.G. Ades S.E. Singer S.J. Hynes R.O. Nature. 1990; 347: 685-689Crossref PubMed Scopus (240) Google Scholar, 22Hamada K. Shimizu T. Matsui T. Tsukita S. Tsukita S. Hakoshima T. EMBO J. 2000; 19: 4449-4462Crossref PubMed Scopus (310) Google Scholar), and has binding sites for the cytoplasmic domains of β1A, β1D (23Calderwood D.A. Zent R. Grant R. Rees D.J.G. Hynes R.O. Ginsburg M.H. J. Biol. Chem. 1999; 274: 28071-28704Abstract Full Text Full Text PDF PubMed Scopus (552) Google Scholar), and β3 (24Pfaff M. Liu S. Erle D.J. Ginsberg M.H. J. Biol. Chem. 1998; 273: 6104-6109Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar) integrins (K d = ∼100 nm) (25Yan B. Calderwood D.A. Yaspan B. Ginsberg M.H. J. Biol. Chem. 2001; 276: 28164-28170Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar) as well as the C-type lectin layilin Hynes R.O. J. Cell Biol. 1998; PubMed Scopus Google Scholar), the kinase Hynes R.O. J. Cell Biol. 1998; PubMed Scopus Google Scholar), and F-actin L. Rees D.J.G. Ohanian V. Bolton S.J. Gilmore A.P. Patel N. Priddle H. Trevithick J.E. Hynes R.O. Critchley D.R. J. Cell Sci. 1996; 109: 2715-2726Crossref PubMed Google Scholar). The talin which is for the of the talin anti-parallel M. S. Y. T. S. J. Biochem. 1995; PubMed Scopus Google Scholar), contains a highly conserved site (residues L. Rees D.J.G. Ohanian V. Bolton S.J. Gilmore A.P. Patel N. Priddle H. Trevithick J.E. Hynes R.O. Critchley D.R. J. Cell Sci. 1996; 109: 2715-2726Crossref PubMed Google Scholar, R.O. Biochem. Biophys. Res. Commun. 1999; PubMed Scopus Google Scholar), which probably for the actin-cross-linking of It also contains a second site (residues (25Yan B. Calderwood D.A. Yaspan B. 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Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). of the by is an complex also PIP kinase or both integrins and PIP kinase for binding to the same on the talin F3 subdomain. we have used NMR and fluorescence to the interaction between a talin polypeptide and a PIP kinase peptide containing the minimal talin-binding The results that in the talin F3 is in binding both the PIP kinase type 1γ and the Moreover, the of the cytoplasmic domain to bind talin is inhibited by a PIP kinase type 1γ peptide. and of the talin were by a mouse (20Rees D.J.G. Ades S.E. Singer S.J. Hynes R.O. Nature. 1990; 347: 685-689Crossref PubMed Scopus (240) Google Scholar) and into the and sites of the expression 15N-labeled proteins were expressed as by M. C. J. 2001; PubMed Scopus Google Scholar) or were with or The proteins were using a and with The was by the of and the protein was using a exchange by using a in of the proteins were and to on the of the NMR of Talin to PIP by NMR and PIP kinase peptide containing the minimal talin-binding site L. K. R. S. S. J. M.R. P. Nature. 2002; PubMed Scopus Google Scholar) was and the by of was by The protein and peptide were determined the of the PIP kinase peptide in NMR to talin was using and on a The were and using and were to the were using an or a and were for were using an with and the and as A. R.J. 2000; PubMed Scopus Google Scholar). The were to single to an rate = and the and the rate were of the PIP kinase second by the of Talin were as L. K. R. S. S. J. M.R. P. Nature. 2002; PubMed Scopus Google Scholar). rat brain extracts were on containing of or of the of PIP kinase as well as of the integrin cytodomain A. Peter K. J. Cell Res. 1999; PubMed Scopus Google Scholar) in and with a of four with the same was in and for and or In the brain extracts were for with a PIP kinase peptide containing the minimal talin-binding site L. K. R. S. S. J. M.R. P. Nature. 2002; PubMed Scopus Google Scholar) and a control peptide of the Talin FERM by of of the complete talin FERM domain residues the F2F3 (residues and the (residues is in The of the is the of and of an and highly region a and region is to the because the of F2F3 or not have and probably the to in on K. Shimizu T. Matsui T. Tsukita S. Tsukita S. Hakoshima T. EMBO J. 2000; 19: 4449-4462Crossref PubMed Scopus (310) Google Scholar). The of regions in the F2F3 and that of the FERM domain expressed a globular structure that the and Moreover, of the of the and F2F3 with that of the complete FERM domain that the of the have the same that the the However, of the not have a the same in the between the of these are consistent with the known of FERM which of three globular by K. Shimizu T. Matsui T. Tsukita S. Tsukita S. Hakoshima T. EMBO J. 2000; 19: 4449-4462Crossref PubMed Scopus (310) Google Scholar). of of the Talin FERM to PIP 1γ by of the PIP kinase peptide in in the of three talin In the of F2F3 and not the exchange between the and the was on the NMR as shown by the of in the the of peptide. The of these increased with peptide and a a 1:1 In a similar number of in and a 1:1 of peptide did not in the and to the of the peptide in the The PIP kinase peptide also in the but the number of and the of the were with the F2F3 polypeptide. Moreover, of with peptide to a shift of the to exchange on the NMR The that the interaction of the PIP kinase peptide with the and F2F3 is that with the and that the number of residues in binding is in the that the F3 contains the binding site for PIP is in complete with the of L. K. R. S. S. J. M.R. P. Nature. 2002; PubMed Scopus Google Scholar), the PIP kinase-binding site to the talin F3 using the same the in the peptide binding may a domain to the binding of the PIP in the Talin F3 of the PIP kinase-binding site within F3 was analysis of the in the of the F2F3 subdomain. we used the well to the Arg were for F2F3 and four for in with the four Arg residues in and three in The of the three Arg residues the F3 are and of the PIP kinase peptide the of two of these of the are not We the F3 Arg to residues using and R358A to the of a single in the the Arg were in to those of the The in that PIP kinase binding the of and for these residues in PIP kinase binding. the R339A exchange on the NMR was similar to that of the the R358A and R358K the exchange between the and the was on the NMR as shown by the of a large number of in the F2F3 a of a of in peptide the of affect of the R339A mutation that is in the of the binding site but is not in of the PIP kinase-binding site was the of The of Ile and Val residues in the F3 crystal structure B. de Pereda J.M. Calderwood D.A. Critchley D.R. Ginsberg M.H. Liddington R.C. Cell. 11: Full Text Full Text PDF PubMed Scopus Google Scholar) is shown in The of Ile residues are in the in the of but on the of the In of Val residues are in the of and We very large chemical shift of the of four of eight and a in the of one of the seven Val the of in the interaction with the peptide. contains Ile residues with and the to to binding as are the of is the and also The Val that has an on the same surface is which with the of peptide binding on the Val of the Ile and Val of the talin F2F3 or the PIP kinase peptide. of the talin F2F3 polypeptide 15N-labeled Ile or Val residues in the and in the of of PIP kinase peptide are of Talin FERM by of a Trp in the PIP kinase peptide to fluorescence to the The of the PIP kinase peptide was of a Trp in an the Trp in the talin of In the of the F2F3 the PIP kinase Trp to a and the fluorescence was by with the of the talin and PIP kinase The of the fluorescence binding was by the of the and rate for the binding were fluorescence on the type F2F3 as well as the Arg and are in The R358K and R358A a large reduction in binding The in for the to the exchange to on the NMR As in the chemical shift between the and the are for to are for the type F2F3 with exchange but are with the for the consistent with exchange and the exchange for the interaction between the PIP kinase peptide and talin F2F3 by fluorescence in a A of the Talin F3 The NMR in the talin F3 PIP kinase binding and the of F3 mutations on the interaction the minimal PIP kinase-binding site in F3 to the in in the surface formed by the and the The same surface been as the binding for the integrin cytodomain by on a talin chimera B. de Pereda J.M. Calderwood D.A. Critchley D.R. Ginsberg M.H. Liddington R.C. Cell. 11: Full Text Full Text PDF PubMed Scopus Google Scholar). to the of the Trp present in the integrin cytodomain to the as one of the of the is into a in the talin F3 formed by the of and The of a with the of and a to that leads to the of the of to markedly binding of talin F2F3 to the β3-integrin cytodomain in B. de Pereda J.M. Calderwood D.A. Critchley D.R. Ginsberg M.H. Liddington R.C. Cell. 11: Full Text Full Text PDF PubMed Scopus Google Scholar). The integrin polypeptide the of the F3 surface formed by the and the B. de Pereda J.M. Calderwood D.A. Critchley D.R. Ginsberg M.H. Liddington R.C. Cell. 11: Full Text Full Text PDF PubMed Scopus Google Scholar) in the of and The in the of the integrin cytodomain the into with in talin The region the N-terminal of the integrin is in with the of binding of the integrin peptide well with on kinase R358A or R358K mutations in F3 the K d for the PIP kinase peptide. The between integrin and PIP kinase binding also to the that both also with the (3Calderwood D.A. Yan B. de Pereda J.M. Alvarez B.G. Fujioka Y. Liddington R.C. Ginsberg M.H. J. Biol. Chem. 2002; 277: 21749-21758Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar). of the PIP kinase peptide with integrin that of integrin residues and to Val and in PIP kinase it more for binding to the present on the talin F3 binding of PIP kinase is Indeed, we failed to binding of a integrin β3 cytodomain peptide is to talin F2F3 either by NMR or by that although the region is it is not for integrin and that regions of the integrin cytodomain as B. A. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). with talin binding results in NMR chemical shift in the membrane-proximal region of the integrin cytodomain A. A. B. J. Cell. 2002; 110: Full Text Full Text PDF PubMed Scopus Google Scholar). of Talin to by a PIP 1γ that the PIP kinase type and the integrin binds to the same or an site in the talin F3 we to a PIP kinase peptide containing a minimal talin-binding site the The results in that talin present in a rat brain binds to a kinase protein and that a PIP kinase peptide containing the a peptide in which the residues were to such PIP kinase were for to binding of talin to a integrin β1-cytodomain Talin present in the brain to the and binding was inhibited by the type PIP kinase peptide but not that containing the to results that the integrin β1-cytodomain binds to the talin FERM domain or to the same site as PIP have shown that an interaction between talin and PIP kinase results in activation of the kinase and of the complex to the K. R.A. Nature. 2002; PubMed Scopus Google Scholar). of the sites in talin V. C. S. C. A. Block M.R. C. J. Biol. 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