A Molecular Mechanism for the Requirement of PAT-4 (Integrin-linked Kinase (ILK)) for the Localization of UNC-112 (Kindlin) to Integrin Adhesion Sites

Qadota, Hiroshi; Moerman, Donald G.; Benian, Guy M.

doi:10.1074/jbc.m112.354852

Cited by 29 publications

(70 citation statements)

References 46 publications

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“…Our previous data support a model in which one of the functions of the interaction of PAT-4 with UNC-112 is to promote opening up of UNC-112 so that it can bind to the cytoplasmic tail of PAT-3 (␤-integrin) (1). In agreement with this model, we have shown here that in vivo, expression of one of the PAT-4 missense mutants permits UNC-112 D382V to localize to integrin adhesion sites (Fig.…”

Section: Discussionsupporting

confidence: 89%

“…Screening for PAT-4 Suppressor Mutations That Can Bind to UNC-112 (D382V)-We utilized random mutagenesis by PCR, as described previously (1). PAT-4 cDNAs with mutations were amplified with two primers (5Ј in (GAA GAT ACC CCA CCA AAC) and 3Ј in (AAA GAA GGC AAA ACG ATG)) and a PAT-4 cDNA plasmid (pDM#280) (13) as a template.…”

Section: Methodsmentioning

confidence: 99%

“…UNC-112 (D382V) was described previously (1). XhoI fragments of PAT-4 cDNA of either wild type or from pACT clones were cloned into the SalI site of pGBDU-C2, resulting in pGBDU-PAT-4 (wild type or P257L).…”

Section: Expression Of Ha-tagged Unc-112 and Myc-tagged Pat-4 In C Ementioning

confidence: 99%

“…Lysates-Nematode-integrated transgenic lines sfIs3 and sfIs5 (1) were induced to express HA-tagged UNC-112 wild type or UNC-112 D382V, respectively, by heat shocking mostly adult animals by exposure to 30°C for 3 h, and worm lysates were prepared as described by Qadota et al (1). To 500 l of each worm lysate was added 50 l of a 1:1 slurry of monoclonal anti-HA-agarose beads (Sigma-Aldrich A2095) and mixing at 4°C for 2 h. The beads were pelleted and washed three times with lysis buffer, transferred to new tubes, pelleted, and as much liquid was removed as possible.…”

Section: Detection Of Complex Formation From Wormmentioning

confidence: 99%

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Suppressor Mutations Suggest a Surface on PAT-4 (Integrin-linked Kinase) That Interacts with UNC-112 (Kindlin)

Qadota

Luo

Matsunaga

et al. 2014

Journal of Biological Chemistry

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Background: Interaction between PAT-4 (ILK) and UNC-112 (kindlin) is required for localization of each protein to integrin adhesions. Results: PAT-4 missense mutations were identified that restore binding of a mutant UNC-112 protein and its proper localization. Conclusion: Residues mutated in the suppressor mutants cluster in two regions on the surface of a homology model of PAT-4. Significance: An interaction surface between PAT-4 and UNC-112 is suggested.

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Section: Discussionsupporting

confidence: 89%

Section: Methodsmentioning

confidence: 99%

Section: Expression Of Ha-tagged Unc-112 and Myc-tagged Pat-4 In C Ementioning

confidence: 99%

Section: Detection Of Complex Formation From Wormmentioning

confidence: 99%

See 2 more Smart Citations

Suppressor Mutations Suggest a Surface on PAT-4 (Integrin-linked Kinase) That Interacts with UNC-112 (Kindlin)

Qadota

Luo

Matsunaga

et al. 2014

Journal of Biological Chemistry

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“…4D). This is consistent with yeast two-hybrid data (Mackinnon et al, 2002;Qadota et al, 2012) and strongly suggests that the kindlin-2 F2PH region mediates a direct interaction with the ILK kinase domain.…”

Section: Kindlin-2 F2ph Directly Interacts With the Kinase Domain Of Ilksupporting

confidence: 90%

Differential binding to the ILK complex determines kindlin isoform adhesion localization and integrin activation

Huet-Calderwood

Brahme

Kumar

et al. 2014

Journal of Cell Science

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BSTRACTKindlins are essential FERM-domain-containing focal adhesion (FA) proteins required for proper integrin activation and signaling. Despite the widely accepted importance of each of the three mammalian kindlins in cell adhesion, the molecular basis for their function has yet to be fully elucidated, and the functional differences between isoforms have generally not been examined. Here, we report functional differences between kindlin-2 and -3 (also known as FERMT2 and FERMT3, respectively); GFP-tagged kindlin-2 localizes to FAs whereas kindlin-3 does not, and kindlin-2, but not kindlin-3, can rescue a5b1 integrin activation defects in kindlin-2-knockdown fibroblasts. Using chimeric kindlins, we show that the relatively uncharacterized kindlin-2 F2 subdomain drives FA targeting and integrin activation. We find that the integrin-linked kinase (ILK)-PINCH-parvin complex binds strongly to the kindlin-2 F2 subdomain but poorly to that of kindlin-3. Using a point-mutated kindlin-2, we establish that efficient kindlin-2-mediated integrin activation and FA targeting require binding to the ILK complex. Thus, ILK-complex binding is crucial for normal kindlin-2 function and differential ILK binding contributes to kindlin isoform specificity.

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The binding interface of kindlin‐2 and ILK involves Asp344/Asp352/Thr356 in kindlin‐2 and Arg243/Arg334 in ILK

et al. 2018

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Focal adhesion (FA) proteins, kindlin-2 and integrin-linked kinase (ILK), regulate cell adhesion and migration. ILK interacts with and promotes kindlin-2 targeting to FAs. Leu353 and Leu357 in kindlin-2 have been reported to be important for the interaction between kindlin-2 and ILK. However, the binding interface between kindlin-2 and ILK remains unclear. Using molecular modeling and molecular dynamics simulations, we show that Asp344, Asp352, and Thr356 in kindlin-2 and Arg243 and Arg334 in ILK kinase domain (KD) are important in kindlin-2/ILK complex formation. Mutations that disrupt these interactions abrogate kindlin-2 and ILK colocalization in HeLa cells. The interactions are direct based on data from pull-down assays using purified recombinant kindlin-2 F2-pleckstrin homology and ILK KDs. These data provide additional insights into the binding interface between kindlin-2 and ILK.

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A Molecular Mechanism for the Requirement of PAT-4 (Integrin-linked Kinase (ILK)) for the Localization of UNC-112 (Kindlin) to Integrin Adhesion Sites

Cited by 29 publications

References 46 publications

Suppressor Mutations Suggest a Surface on PAT-4 (Integrin-linked Kinase) That Interacts with UNC-112 (Kindlin)

Suppressor Mutations Suggest a Surface on PAT-4 (Integrin-linked Kinase) That Interacts with UNC-112 (Kindlin)

Differential binding to the ILK complex determines kindlin isoform adhesion localization and integrin activation

The binding interface of kindlin‐2 and ILK involves Asp344/Asp352/Thr356 in kindlin‐2 and Arg243/Arg334 in ILK

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