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• W3128148207 abstract "Integrin-linked kinase (ILK), a central component of the intracellular ILK–pinch–parvin complex, localizes together with paxillin to focal adhesions and regulates integrin-mediated cell functions. ILK was initially misclassified as a kinase based on phenotypical characterization of cells expressing ILK mutated in the “kinase” domain, such as the E359K and K220M mutants and a V386G/T387G mutation in the paxillin-binding site (PBS). ILK is now known to be a pseudokinase, and mechanisms of action of these mutants are not clear. We selectively induced expression of only the E359K, PBS, and K220M ILK mutations in the developing kidney collecting system and kidney collecting duct (CD) cells and analyzed their impact on structural integrity using molecular dynamics (MD) simulations. Mice or CD cells carrying the E359K mutation had a severe phenotype that is indistinguishable from ILK-null mice or ILK-null CD cells. The K220M mutant mice developed normally, and K220M-CD cells had a mild adhesion, migration, and tubulogenesis defect. The PBS mutant mice had a subtle developmental defect, and PBS-CD cells had moderate functional abnormalities. Consistent with these observed phenotypes, MD studies suggest that the E359K mutant produces the most structurally perturbed, and K220M the most WT-like ILK molecules. Although all three mutations disrupted ILK binding to parvin and paxillin in vitro, only the E359K mutation decreased ILK binding to pinch suggesting that it increases ILK misfolding. Thus, point mutations in the ILK pseudokinase domain cause functional abnormalities by altering the ILK structure, leading to increased turnover and destabilization of ILK–parvin and (sometimes) ILK–pinch interactions. The integrin-linked kinase (ILK)–pinch–parvin (IPP) complex is a critical component of focal adhesions that binds to the cytoplasmic tail of the integrin β subunits. Integrins, composed of an α and a β subunit, are the principal receptors that mediate cell–extracellular matrix interactions and regulate many cell functions, including adhesion, spreading, migration, polarization, and tubulogenesis. ILK is a 450 amino acid multidomain pseudokinase protein consisting of an N-terminal domain with five ankyrin repeats, a COOH-terminal pseudo kinase domain, and an intervening pleckstrin homology (PH) domain (1Ghatak S. Morgner J. Wickstrom S.A. ILK: A pseudokinase with a unique function in the integrin-actin linkage.Biochem. Soc. Trans. 2013; 41: 995-1001Crossref PubMed Scopus (53) Google Scholar, 2Wickstrom S.A. Lange A. Montanez E. Fassler R. The ILK/PINCH/parvin complex: The kinase is dead, long live the pseudokinase!.EMBO J. 2010; 29: 281-291Crossref PubMed Scopus (181) Google Scholar, 3Fukuda K. Gupta S. Chen K. Wu C. Qin J. The pseudoactive site of ILK is essential for its binding to alpha-Parvin and localization to focal adhesions.Mol. Cell. 2009; 36: 819-830Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 4Qin J. Wu C. ILK: A pseudokinase in the center stage of cell-matrix adhesion and signaling.Curr. Opin. Cell Biol. 2012; 24: 607-613Crossref PubMed Scopus (81) Google Scholar). Pinch interacts with the ankyrin repeat domain, while parvin family members bind to the pseudokinase domain. The three components of the IPP complex are thought to assemble in the cytosol, and the pseudokinase domain is required for this interaction to occur. The pseudokinase domain is inherently unstable, and the heat shock protein Hsp90 is required to stabilize interactions between parvin and ILK (5Wickstrom S.A. Lange A. Hess M.W. Polleux J. Spatz J.P. Kruger M. Pfaller K. Lambacher A. Bloch W. Mann M. Huber L.A. Fassler R. Integrin-linked kinase controls microtubule dynamics required for plasma membrane targeting of caveolae.Dev. Cell. 2010; 19: 574-588Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). The expression of all three IPP components is critical for IPP complex formation as genetic deletion of either pinch or ILK leads to a decrease in IPP complex formation; however, the mechanism for this interdependency is unknown (2Wickstrom S.A. Lange A. Montanez E. Fassler R. The ILK/PINCH/parvin complex: The kinase is dead, long live the pseudokinase!.EMBO J. 2010; 29: 281-291Crossref PubMed Scopus (181) Google Scholar). ILK is critical for survival in mice and its constitutive loss results in lethality at the periimplantation stage due to abnormal epiblast polarity and adhesion (6Sakai T. Li S. Docheva D. Grashoff C. Sakai K. Kostka G. Braun A. Pfeifer A. Yurchenco P.D. Fassler R. Integrin-linked kinase (ILK) is required for polarizing the epiblast, cell adhesion, and controlling actin accumulation.Genes Dev. 2003; 17: 926-940Crossref PubMed Scopus (310) Google Scholar). Variable phenotypes occur with tissue-specific deletion of ILK, which causes branching morphogenesis defects in the developing kidney collecting system (7Smeeton J. Zhang X. Bulus N. Mernaugh G. Lange A. Karner C.M. Carroll T.J. Fassler R. Pozzi A. Rosenblum N.D. Zent R. Integrin-linked kinase regulates p38 MAPK-dependent cell cycle arrest in ureteric bud development.Development. 2010; 137: 3233-3243Crossref PubMed Scopus (32) Google Scholar) and mammary gland (8Akhtar N. Marlow R. Lambert E. Schatzmann F. Lowe E.T. Cheung J. Katz E. Li W. Wu C. Dedhar S. Naylor M.J. Streuli C.H. Molecular dissection of integrin signalling proteins in the control of mammary epithelial development and differentiation.Development. 2009; 136: 1019-1027Crossref PubMed Scopus (53) Google Scholar). A point mutagenesis strategy was employed to define the mechanisms of ILK function in the setting of the IPP complex. Mice carrying a constititve and homozygous R211A or the S343A/D mutations in the pseudokinase domain are normal, while mice with a K220A/M mutation in the same domain show kidney developmental abnormalities due to decreased interactions between ILK and α-parvin caused by increased destabilization of the ILK pseudokinase domain structure (9Lange A. Wickstrom S.A. Jakobson M. Zent R. Sainio K. Fassler R. Integrin-linked kinase is an adaptor with essential functions during mouse development.Nature. 2009; 461: 1002-1006Crossref PubMed Scopus (97) Google Scholar). ILK has also been proposed to bind directly to the cytosolic signaling protein paxillin via a highly conserved paxillin-binding site (PBS) found in the pseudokinase domain of ILK as well as several other proteins (10Nikolopoulos S.N. Turner C.E. Integrin-linked kinase (ILK) binding to paxillin LD1 motif regulates ILK localization to focal adhesions.J. Biol. Chem. 2001; 276: 23499-23505Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 11Nikolopoulos S.N. Turner C.E. Molecular dissection of actopaxin-integrin-linked kinase-Paxillin interactions and their role in subcellular localization.J. Biol. Chem. 2002; 277: 1568-1575Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 12Moik D. Bottcher A. Makhina T. Grashoff C. Bulus N. Zent R. Fassler R. Mutations in the paxillin-binding site of integrin-linked kinase (ILK) destabilize the pseudokinase domain and cause embryonic lethality in mice.J. Biol. Chem. 2013; 288: 18863-18871Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). Mice carrying the V386G/T387G mutation in the PBS-binding sites die at E9.5, and in vitro studies revealed that these mutations alter ILK stability and decrease its ability to bind α-parvin or paxillin. Finally, an E359K mutation was made based on a Glu-Arg salt bridge that connects coevolved motifs that defines the highly conserved fold found in in all eukarytoic protein kinases (13Yang J. Wu J. Steichen J.M. Kornev A.P. Deal M.S. Li S. Sankaran B. Woods Jr., V.L. Taylor S.S. A conserved Glu-Arg salt bridge connects coevolved motifs that define the eukaryotic protein kinase fold.J. Mol. Biol. 2012; 415: 666-679Crossref PubMed Scopus (30) Google Scholar). However, it is now clear that ILK is not a kinase and the mechanism of action of this mutant deviates from structurally homologus eukaryotic protein kinases, thus its effects in vivo must now be regarded as unknown (11Nikolopoulos S.N. Turner C.E. Molecular dissection of actopaxin-integrin-linked kinase-Paxillin interactions and their role in subcellular localization.J. Biol. Chem. 2002; 277: 1568-1575Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 14Leung-Hagesteijn C. Hu M.C. Mahendra A.S. Hartwig S. Klamut H.J. Rosenblum N.D. Hannigan G.E. Integrin-linked kinase mediates bone morphogenetic protein 7-dependent renal epithelial cell morphogenesis.Mol. Cell Biol. 2005; 25: 3648-3657Crossref PubMed Scopus (42) Google Scholar, 15Miller M.G. Naruszewicz I. Kumar A.S. Ramlal T. Hannigan G.E. Integrin-linked kinase is a positive mediator of L6 myoblast differentiation.Biochem. Biophys. Res. Commun. 2003; 310: 796-803Crossref PubMed Scopus (22) Google Scholar, 16Levinson H. Turner C.E. Ehrlich H.P. Integrin-linked kinase: A possible role in scar contracture.Ann. Plast. Surg. 2004; 52: 204-211Crossref PubMed Scopus (5) Google Scholar, 17Hwangbo C. Park J. Lee J.H. mda-9/Syntenin protein positively regulates the activation of Akt protein by facilitating integrin-linked kinase adaptor function during adhesion to type I collagen.J. Biol. Chem. 2011; 286: 33601-33612Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). We previously showed that deleting ILK in the developing ureteric bud (UB) of the kidney results in a branching morphogenesis defect and that ILK-null collecting duct (CD) cells have abnormalities in multiple integrin-dependent functions (7Smeeton J. Zhang X. Bulus N. Mernaugh G. Lange A. Karner C.M. Carroll T.J. Fassler R. Pozzi A. Rosenblum N.D. Zent R. Integrin-linked kinase regulates p38 MAPK-dependent cell cycle arrest in ureteric bud development.Development. 2010; 137: 3233-3243Crossref PubMed Scopus (32) Google Scholar). In this study, we generated mutations in the developing UB and CD cells to define the mechanisms whereby K220M, E359K, and PBS mutations alter ILK function in the setting of polarized epithelium. We subsequently employed molecular dynamic (MD) simulations and molecular modeling techniques to visualize how these ILK mutations can disrupt the IPP complex. ILK is required for normal development, and global ILK-null mice die at the periimplantation stage (6Sakai T. Li S. Docheva D. Grashoff C. Sakai K. Kostka G. Braun A. Pfeifer A. Yurchenco P.D. Fassler R. Integrin-linked kinase (ILK) is required for polarizing the epiblast, cell adhesion, and controlling actin accumulation.Genes Dev. 2003; 17: 926-940Crossref PubMed Scopus (310) Google Scholar). Mice constitutively expressing K220M die shortly after birth from kidney development complications (9Lange A. Wickstrom S.A. Jakobson M. Zent R. Sainio K. Fassler R. Integrin-linked kinase is an adaptor with essential functions during mouse development.Nature. 2009; 461: 1002-1006Crossref PubMed Scopus (97) Google Scholar), and PBS ILK mutants do not survive longer than E9.5 (12Moik D. Bottcher A. Makhina T. Grashoff C. Bulus N. Zent R. Fassler R. Mutations in the paxillin-binding site of integrin-linked kinase (ILK) destabilize the pseudokinase domain and cause embryonic lethality in mice.J. Biol. Chem. 2013; 288: 18863-18871Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). We generated the constitutive E359K mutant mice as described in the Experimental procedures and Figure S1 and found that they have an early embryonic lethal phenotype (data not shown). To compare the differential effects of the E359K, K220M, and PBS mutations in the same biological system in vivo, we expressed the mutant ILK versions in the UB, which gives rise to the kidney collecting system. This was achieved by intercrossing the heterozygous E359K, K220M, and PBS mutant mice with ILK-floxed mice expressing Cre recombinase under the hoxB7 promoter (18Yu J. Carroll T.J. McMahon A.P. Sonic hedgehog regulates proliferation and differentiation of mesenchymal cells in the mouse metanephric kidney.Development. 2002; 129: 5301-5312Crossref PubMed Google Scholar). The heterozygous mutant mice and heterozygous floxed ILK mice crossed with hoxB7 Cre mice were normal (data not shown). The breeding strategy used should result in 12.5% of the offspring expressing the mutant protein in the UB. Littermates carrying the floxed and mutated ILK alleles without the Cre recombinase transgene served as controls for experiments. The PBS and K220M mutant mice were born in the correct Mendelian ratio while the E359K mutants were born at a lower ratio (6.7%), which was similar to the mice lacking ILK in the UB (8.1%), suggesting that some of these mice died in utero or at birth. The PBS and K220M mice developed normally and did not display overt developmental phenotypes. By contrast, the surviving E359K and the UB-ILK null mice died at about 8 weeks of age from renal failure. We analyzed kidneys from 1-year-old PBS and K220M mice and the E359K mice just prior to death (Fig. 1, A–D). The K220M kidneys were normal and indistinguishable from control mice at all time points analyzed. The PBS mutant kidneys displayed a subtle branching morphogenesis defect characterized by less tubules in the kidney papilla (Fig. 1C). By contrast, the E359K mice either developed obstruction or the kidneys were severely dysplastic and hypoplastic (Fig. 1B), consistent with defects seen in the UB-ILK-null mice (7Smeeton J. Zhang X. Bulus N. Mernaugh G. Lange A. Karner C.M. Carroll T.J. Fassler R. Pozzi A. Rosenblum N.D. Zent R. Integrin-linked kinase regulates p38 MAPK-dependent cell cycle arrest in ureteric bud development.Development. 2010; 137: 3233-3243Crossref PubMed Scopus (32) Google Scholar). Abnormalities in the E359K kidneys were evident at all embryological developmental stages, and at E15.5 the kidneys were noted to be small with a moderate branching morphogenesis defect and impaired metanephric mesenchyme induction (Fig. 1, E and F). Kidneys at P0 were hypoplastic and dysplastic (Fig. 1, G and H), and they became dilated over time due to the intraluminal obstruction in the collecting ducts (Fig. 1, I–L). During the postnatal period, the tubules did not have any cell survival defects, but displayed intratubular proliferation characterized by increased Ki67 cells (Fig. 1, M–O), similar to that seen in UB-ILK-null mice (7Smeeton J. Zhang X. Bulus N. Mernaugh G. Lange A. Karner C.M. Carroll T.J. Fassler R. Pozzi A. Rosenblum N.D. Zent R. Integrin-linked kinase regulates p38 MAPK-dependent cell cycle arrest in ureteric bud development.Development. 2010; 137: 3233-3243Crossref PubMed Scopus (32) Google Scholar). Thus, introducing the K220 M mutation does not cause any detectable developmental abnormalities in the collecting system, and the PBS mutation causes a subtle nonprogressive branching defect with no evidence of obstruction. On the other hand, the E359K mutation causes a severe developmental phenotype that is indistinguishable from the UB-ILK null mice (7Smeeton J. Zhang X. Bulus N. Mernaugh G. Lange A. Karner C.M. Carroll T.J. Fassler R. Pozzi A. Rosenblum N.D. Zent R. Integrin-linked kinase regulates p38 MAPK-dependent cell cycle arrest in ureteric bud development.Development. 2010; 137: 3233-3243Crossref PubMed Scopus (32) Google Scholar). To define the mechanisms of action of the various ILK mutants in polarized renal epithelial cells, we generated stable clones of ILK-null CD cells (7Smeeton J. Zhang X. Bulus N. Mernaugh G. Lange A. Karner C.M. Carroll T.J. Fassler R. Pozzi A. Rosenblum N.D. Zent R. Integrin-linked kinase regulates p38 MAPK-dependent cell cycle arrest in ureteric bud development.Development. 2010; 137: 3233-3243Crossref PubMed Scopus (32) Google Scholar) expressing comparable levels of wild-type (WT) ILK and the three ILK mutants (Fig. 2A). Multiple clones were generated and shown to have similar phenotypes. We initially performed branching morphogenesis assays in three-dimensional collagen I/Matrigel gels. CD cells expressing WT-ILK were able to form tubules with lumens (arrow) that were visible in single-plane confocal pictures (Fig. 2B). By contrast, the E359K CD cells were unable to undergo branching morphogenesis and made multicellular aggregates within the gel (Fig. 2, B, C, and F). The PBS formed unicellular cellular outgrowths with no visible lumens and had half as many branched structures per tubule as the WT-ILK cells (Fig. 2, D and F). Although the K220M mutant cells developed multicellular structures with lumens (arrow), they had half the branch number of the WT-ILK cells (Fig. 2, E and F). We next investigated well-described integrin/ILK-mediated functions, which include cell adhesion, haptotactic cell migration, cell proliferation, and spreading on extracellular matrices (Fig. 2, G–N). In all these functional assays, the E359K-CD cells had the most severe defects when plated on either collagen I or Matrigel. The PBS mutant cells showed less severe, yet significant defects, while the K220M cells showed the least severe phenotype with a mild yet significant adhesion and migration defect, but no differences in proliferation or spreading when compared with cells expressing WT-ILK (Fig. 2, G–N). Together these results demonstrate that the three mutations within the pseudokinase domain of ILK variably affect multiple integrin-dependent cell functions. We next investigated the mechanisms whereby the mutants induce their different phenotypes in CD cells. We initially defined the effects of the various ILK mutants on the levels of the major components of the IPP complex and paxillin, which is thought to bind to ILK via the highly conserved PBS site. Immunoblots of total cell lysates of CD cells expressing WT-ILK and the K220M mutation demonstrated that all three components of the IPP complex as well as paxillin were present (Fig. 3, A and B); however, similar to cells lacking ILK (KO), there was almost no α-parvin in the lysates of the E359K and PBS mutants (Fig. 3, A and B). Similar results were observed for β-parvin, which is also expressed in CD cells (Fig. S2), demonstrating that there is no compensation by β-parvin for the decreased α-parvin. There were no differences in the amount of pinch or paxillin in cells expressing WT-ILK or any of the mutants (Fig. 3, A and B). We next defined which proteins formed a complex with ILK by performing 12 h immunoprecipitation assays with an antibody directed against FLAG. Similar amounts of ILK were immunoprecipitated from WT- and K220M-ILK expressing cells; however, less was immunoprecipitated from the PBS and E359K mutants, respectively, suggesting there may be degradation of these mutant forms of ILK. Almost no α-parvin or β-parvin, pinch, or paxillin was immunoprecipitated with the E359K mutant (Fig. 3, C and D and Fig. S2). By contrast, pinch, but not α-parvin, β-parvin, or paxillin, was coimmunoprecipitated with the PBS or K220M mutants. We next examined whether the mutant ILK proteins localize to focal adhesion on cells that were plated on collagen I by performing costaining with ILK and phospho-paxillin antibodies. Interestingly, the WT-ILK as well as PBS and K220M mutants colocalized with phospho-paxillin (Fig. 3, E and F); however, there was almost no colocalization between the E359K mutant and phospho-paxillin, except in large paxillin focal adhesions (Fig. 3, E and F). Thus, E359K-ILK is absent from most focal adhesions and does not form a complex with parvin, paxillin, and pinch. By contrast, the PBS and K220M mutants bind with pinch and localize to focal adhesions even though they do not form a complex with paxillin or parvin. We previously showed that the cellular lifetime of the PBS mutant is decreased relative to WT-ILK (12Moik D. Bottcher A. Makhina T. Grashoff C. Bulus N. Zent R. Fassler R. Mutations in the paxillin-binding site of integrin-linked kinase (ILK) destabilize the pseudokinase domain and cause embryonic lethality in mice.J. Biol. Chem. 2013; 288: 18863-18871Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). We therefore dermined WT, E359K-, PBS-, and K220M-ILK levels in CD cells 8, 18, and 24 h after treatment with cylohexamide (to inhibit protein synthesis). There was an approximately 20% decrease of WT ILK at 8 h, which stayed relatively constant until the 24 h time point. By contrast, the amount of E359K decreased by about 30% at 8 h, 50% at 18 h, and 68% at 24 h. The decrease in the PBS and K220M mutants was between the WT and E359K mutants at 18 and 24 h (Fig. 4, A and B). The half-lives of WT, E359K-, PBS-, and K220M-ILK are 63.7, 15.5, 24.1, and 24.7 h, respectively. As the difference in amounts between all the mutants and WT ILK and between the E359K mutant and the PBS or K220M mutant was significant at 24 h, we assessed the amounts of α-parvin, pinch, and paxillin in the various cell lines at this time point. Cycloheximide decreased α-parvin by approximately 60% in the CD cells expressing WT-ILK, 75% in the E359K and PBS mutants, and 90% in the K220M mutants (Fig. 4, C and D). Interestingly, cycloheximide decreased pinch by 30% in WT and K220M ILK cells, but this was markedly decreased by 90% and 80% in the E359K and PBS mutants, respectively (Fig. 4, C and D). Finally, cycloheximide decreased paxillin by 30% in ILK WT CD cells, and it was down by 50%, 60%, and 70% in the E359K, PBS, and K220M CD cells, respectively (Fig. 4, C and D). Thus, E359K-ILK was turned over quicker than WT-ILK or the other two ILK mutants. Furthermore, α-parvin and pinch turnover was the highest in this E359K mutant followed by the PBS mutant, while in the K220M mutant α-parvin was primarily decreased. Cellular ILK levels are regulated by the Hsp90–Hsc70 chaperone machinery where Hsp90 binds the pseudokinase domain of ILK (19Radovanac K. Morgner J. Schulz J.N. Blumbach K. Patterson C. Geiger T. Mann M. Krieg T. Eckes B. Fassler R. Wickstrom S.A. Stabilization of integrin-linked kinase by the Hsp90-CHIP axis impacts cellular force generation, migration and the fibrotic response.EMBO J. 2013; 32: 1409-1424Crossref PubMed Scopus (47) Google Scholar). Therefore, we tested whether the increased ILK turnover in the E359K, PBS, and K220M mutants was due to a disruption between the mutated ILK proteins and Hsp90. When we immunoprecipitated Hsp90α from the ILK-WT, E359K-, PBS-, and K220M-CD cells and immunoblotted the immunoprecipitated proteins with ILK antibody, ILK was detected in all the cell populations (Fig. 4E). When we immunoprecipitated α-parvin in WT-ILK expressing CD cells, both ILK and Hsp90 were in the immunoprecipitated proteins; however, as expected based on the results shown in Figure 3D, this interaction did not occur in the mutants, where α-parvin is not part of the IPP complex (Fig. 4F). Thus, the mutations in ILK do not alter its ability to bind Hsp-90, which suggests that the increased turnover of the ILK mutants is not due to altered interactions between ILK and the Hsp90–Hsc70 chaperone machinery. As E359K-ILK turnover was significantly higher than either WT-ILK, PBS, and K220M mutants, we tested whether the severe phenotype of this mutant was due to insufficient expression in the cells. We generated CD cells that expressed comparable amounts of WT-ILK and the E359K mutant (E359K/Lo) as well as CD cells that express 2.5 times more E359K-ILK (E359K/Hi) compared with WT-ILK cells (Fig. 5, A and B). In contrast to cells expressing WT-ILK, neither of the E359K mutants expressed α-parvin (Fig. 5A). When adhesion, migration, and proliferation assays were performed, we observed that although E359K/Hi CD cells adhered significantly more than the E359K/Lo CD cells to collagen; they still adhered significantly less than cells expressing WT-ILK (Fig. 5C). The E359K/Hi CD cells did not migrate or proliferate more than the E359K/Lo CD cells (Fig. 5, D and E). Thus, the functional defects observed in cells expressing E359K-ILK are likely due to disruption of global ILK structure that renders it unable to bind to either parvin or pinch. Our results suggest that the E359K, PBS, and K220M mutations caused increased ILK turnover, decreased the amount of parvin (E359K and PBS mutations), and resulted in an inability to form an IPP complex. To better understand the mechanism, we utilized MD simulations of each individual mutation derived from starting coordinates of the WT ILK α-parvin crystal structure (Fig. 6A) (3Fukuda K. Gupta S. Chen K. Wu C. Qin J. The pseudoactive site of ILK is essential for its binding to alpha-Parvin and localization to focal adhesions.Mol. Cell. 2009; 36: 819-830Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). In total, 100 ns trajectories were analyzed to determine the impact of each mutation on global structural features as well as hydrogen bonding networks relative to the WT trajectory. The E359K mutation is located in the APE motif of the putative kinase domain, which forms a conserved salt-bridge with R436 within the GHI-subdomain, which comprises the short G, H, and I helices, (Fig. 6B) with a hydrogen occupancy of 97% and 94% for the E359:OE1-R435:H12 and E359:OE2-R435:H22 bonds, respectively. ILK homology with other eurokeyotic protein kinases (EPKs) indicates that E359 is part of the activation segment found in canonical kinases. In addition, the GHI subdomain is an integral element of the EPK allosteric network that contains the R436 residue of the salt bridge (13Yang J. Wu J. Steichen J.M. Kornev A.P. Deal M.S. Li S. Sankaran B. Woods Jr., V.L. Taylor S.S. A conserved Glu-Arg salt bridge connects coevolved motifs that define the eukaryotic protein kinase fold.J. Mol. Biol. 2012; 415: 666-679Crossref PubMed Scopus (30) Google Scholar). The E359K mutation results in disruption of this highly stable and well-conserved salt bridge as there is now minimal hydrogen binding (<5%) between the lysine sidechain and local backbone oxygens (20Kim C. Cheng C.Y. Saldanha S.A. Taylor S.S. PKA-I holoenzyme structure reveals a mechanism for cAMP-dependent activation.Cell. 2007; 130: 1032-1043Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar) (Fig. 6B). These results suggest that an E359K mutation significantly impacts the ILK molecule's structural stability that impacts multiple possible downstream effects. Paxillin binding is reported to be disrupted in the PBS mutant (10Nikolopoulos S.N. Turner C.E. Integrin-linked kinase (ILK) binding to paxillin LD1 motif regulates ILK localization to focal adhesions.J. Biol. Chem. 2001; 276: 23499-23505Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 11Nikolopoulos S.N. Turner C.E. Molecular dissection of actopaxin-integrin-linked kinase-Paxillin interactions and their role in subcellular localization.J. Biol. Chem. 2002; 277: 1568-1575Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 12Moik D. Bottcher A. Makhina T. Grashoff C. Bulus N. Zent R. Fassler R. Mutations in the paxillin-binding site of integrin-linked kinase (ILK) destabilize the pseudokinase domain and cause embryonic lethality in mice.J. Biol. Chem. 2013; 288: 18863-18871Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). Although paxillin was not included in the simulations, the MD trajectory predicts that the T387 OG1 side-chain oxygen forms hydrogen bonds with the E389 backbone hydrogen (71% occupancy) and with the W383 backbone oxygen (<5% occupancy, Fig. 6C) in the WT system. The PBS mutation replaces V386 and T387 with glycines, which disrupt the hydrophobic interactions of the Val side chain and hydrogen bonds mediated by the Thr hydroxyl side chain. Of interest, a backbone hydrogen bond between the carboxyl oxygen of W383 and amide proton of G387(W383:O-G387:H) was not observed during the PBS MD trajectory. It seems unlikely that loss of this hydrogen bond would yield a global effect on ILK structure. Rather, in conjunction with less favorable hydrophobic interactions with V386G, the loss of the T387G intrahelix hydrogen bond almost certainly disrupts the local stability of the small helix comprising the paxillin binding site and likely affects the overall thermal stability of the ILK molecule. Although ILK has no kinase activity (3Fukuda K. Gupta S. Chen K. Wu C. Qin J. The pseudoactive site of ILK is essential for its binding to alpha-Parvin and localization to focal adhesions.Mol. Cell. 2009; 36: 819-830Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar), it requires ATP for its cellular function (21Vaynberg J. Fukuda K. Lu F. Bialkowska K. Chen Y. Plow E.F. Qin J. Non-catalytic signaling by pseudokinase ILK for regulating cell adhesion.Nat. Commun. 2018; 9: 4465Crossref PubMed Scopus (26) Google Scholar). In the K220M versus WT simulation, the K220 amide side chain was observed to participate in multiple hydrogen bonding with the α- and γ-phosphate oxygen atoms on the bound ATP molecule. Each phosphate group has multiple oxygen atoms that may interact with the K220 sidechain. The sums of the α-phosphate hydrogen bond occupancies with K220:NZH were 62% and 56% for the γ-phosphate; no interactions were observed between K220 and the β-phosphate of ATP. The K220M mutation replaces the electropositive lysine side chain with a nonpolar methionine that does not hydrogen bond with any atoms on the ATP molecule (Fig. 6D). A complete list of hydrogen-bonding occupancies over 10% is provided in Figure S3. Earlier studies show that K220M can alter ILK thermostability (22Fukuda K. Bledzka K. Yang J. Perera H.D. Plow E.F. Qin J. Molecular basis of kindlin-2 binding to integrin-linked kinase pseudokinase for regulating cell adhesion.J. Biol. Chem. 2014; 289: 28363-28375Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Our results predict that K220M directly impairs ATP binding. Although ILK is not a kinase as defined by ATP hyd" @default.
• W3128148207 created "2021-02-15" @default.
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• W3128148207 title "Disruption of the integrin-linked kinase (ILK) pseudokinase domain affects kidney development in mice" @default.
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