Disruption of the integrin-linked kinase (ILK) pseudokinase domain affects kidney development in mice.

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

Talin regulates integrin beta 1-dependent and -independent cell functions in ureteric bud development

Kidney collecting system development requires integrin-dependent cell-extracellular matrix interactions. Integrins are heterodimeric transmembrane receptors consisting of alpha and beta subunits; crucial integrins in the kidney collecting systemexpress the beta 1 subunit. The beta 1 cytoplasmic tail has two NPxYmotifs that mediate functions by binding to cytoplasmic signaling and scaffolding molecules. Talins, scaffolding proteins that bind to the membrane proximal NPxY motif, are proposed to activate integrins and to link them to the actin cytoskeleton. We have defined the role of talin binding to the beta 1 proximal NPxY motif in the developing kidney collecting system in mice that selectively express a Y-to-A mutation in this motif. The mice developed a hypoplastic dysplastic collecting system. Collecting duct cells expressing this mutation had moderate abnormalities in cell adhesion, migration, proliferation and growth factor-dependent signaling. In contrast, mice lacking talins in the developing ureteric bud developed kidney agenesis and collecting duct cells had severe cytoskeletal, adhesion and polarity defects. Thus, talins are essential for kidney collecting duct development through mechanisms that extend beyond those requiring binding to the beta 1 integrin subunit NPxY motif

Integrin-mediated type II TGF-beta receptor tyrosine dephosphorylation controls SWIAD-dependent profibrotic signaling

Tubulointerstitial fibrosis underlies all forms of end-stage kidney disease. TGF-beta mediates both the development and the progression of kidney fibrosis through binding and activation of the serine/threonine kinase type II TGF-beta receptor (T beta RII), which in turn promotes a T beta RI-mediated SMAD-dependent fibrotic signaling cascade. Autophosphorylation of serine residues within T beta RII is considered the principal regulatory mechanism of T beta RII-induced signaling; however, there are 5 tyrosine residues within the cytoplasmic tail that could potentially mediate T beta RII-dependent SMAD activation. Here, we determined that phosphorylation of tyrosines within the T beta RII tail was essential for SMAD-dependent fibrotic signaling within cells of the kidney collecting duct. Conversely, the T cell protein tyrosine phosphatase (TCPTP) dephosphorylated T beta RII tail tyrosine residues, resulting in inhibition of T beta R-dependent fibrotic signaling. The collagen-binding receptor integrin OM was required for recruitment of TCPTP to the T beta RII tail, as mice lacking this integrin exhibited impaired TCPTP-mediated tyrosine dephosphorylation of T beta RII that led to severe fibrosis in a unilateral ureteral obstruction model of renal fibrosis. Together, these findings uncover a crosstalk between integrin alpha 1 beta 1 and T beta RII that is essential for T beta RII-mediated SMAD activation and fibrotic signaling pathways

Integrin alpha 3 beta 1 regulates kidney collecting duct development via TRAF6-dependent K63-linked polyubiquitination of Akt

The collecting system of the kidney develops from the ureteric bud (UB), which undergoes branching morphogenesis, a process regulated by multiple factors, including integrin-extracellular matrix interactions. The laminin (LM)-binding integrin alpha 3 beta 1 is crucial for this developmental program; however, the LM types and LM/integrin alpha 3 beta 1-dependent signaling pathways are poorly defined. We show that alpha 3 chain-containing LMs promote normal UB branching morphogenesis and that LM-332 is a better substrate than LM-511 for stimulating integrin alpha 3 beta 1-dependent collecting duct cell functions. We demonstrate that integrin alpha 3 beta 1-mediated cell adhesion to LM-332 modulates Akt activation in the developing collecting system and that Akt activation is PI3K independent but requires decreased PTEN activity and K63-linked polyubiquitination. We identified the ubiquitin-modifying enzyme TRAF6 as an interactor with the integrin beta 1 subunit and regulator of integrin alpha 3 beta 1-dependent Akt activation. Finally, we established that the developmental defects of TRAF6- and integrin alpha 3-null mouse kidneys are similar. Thus K63-linked polyubiquitination plays a previously unrecognized role in integrin alpha 3 beta 1-dependent cell signaling required for UB development and may represent a novel mechanism whereby integrins regulate signaling pathways