Integrin-Linked Kinase Is a Functional Mn2+-Dependent Protein Kinase that Regulates Glycogen Synthase Kinase-3β (GSK-3β) Phosphorylation

Integrin-linked kinase (ILK) is a highly evolutionarily conserved, multi-domain signaling protein that localizes to focal adhesions, myofilaments and centrosomes where it forms distinct multi-protein complexes to regulate cell adhesion, cell contraction, actin cytoskeletal organization and mitotic spindle assembly. Numerous studies have demonstrated that ILK can regulate the phosphorylation of various protein and peptide substrates in vitro, as well as the phosphorylation of potential substrates and various signaling pathways in cultured cell systems. Nevertheless, the ability of ILK to function as a protein kinase has been questioned because of its atypical kinase domain.Here, we have expressed full-length recombinant ILK, purified it to >94% homogeneity, and characterized its kinase activity. Recombinant ILK readily phosphorylates glycogen synthase kinase-3 (GSK-3) peptide and the 20-kDa regulatory light chains of myosin (LC(20)). Phosphorylation kinetics are similar to those of other active kinases, and mutation of the ATP-binding lysine (K220 within subdomain 2) causes marked reduction in enzymatic activity. We show that ILK is a Mn-dependent kinase (the K(m) for MnATP is approximately 150-fold less than that for MgATP).Taken together, our data demonstrate that ILK is a bona fide protein kinase with enzyme kinetic properties similar to other active protein kinases

A longitudinal twin and sibling study of the hopeless theory of depression in adolescence and young adulthood

Background Maladaptive cognitive biases such as negative attributional style and hopelessness have been implicated in the development and maintenance of depression. According to the hopelessness theory of depression, hopelessness mediates the association between attributional style and depression. The aetiological processes underpinning this influential theory remain unknown. The current study investigated genetic and environmental influences on hopelessness and its concurrent and longitudinal associations with attributional style and depression across adolescence and emerging adulthood. Furthermore, given high co-morbidity between depression and anxiety, the study investigated whether these maladaptive cognitions constitute transdiagnostic cognitive content common to both internalizing symptoms. Method A total of 2619 twins/siblings reported attributional style (mean age 15 and 17 years), hopelessness (mean age 17 years), and depression and anxiety symptoms (mean age 17 and 20 years). Results Partial correlations revealed that attributional style and hopelessness were uniquely associated with depression but not anxiety symptoms. Hopelessness partially mediated the relationship between attributional style and depression. Hopelessness was moderately heritable (A = 0.37, 95% confidence interval 0.28–0.47), with remaining variance accounted for by non-shared environmental influences. Independent pathway models indicated that a set of common genetic influences largely accounted for the association between attributional style, hopelessness and depression symptoms, both concurrently and across development. Conclusions The results provide novel evidence that associations between attributional style, hopelessness and depression symptoms are largely due to shared genetic liability, suggesting developmentally stable biological pathways underpinning the hopelessness theory of depression. Both attributional style and hopelessness constituted unique cognitive content in depression. The results inform molecular genetics research and cognitive treatment approaches

Current understanding of the human microbiome

Author Posting. © The Author(s), 2018. This is the author's version of the work. It is posted here by permission of Nature Publishing Group for personal use, not for redistribution. The definitive version was published in Nature Medicine 24 (2018): 392–400, doi:10.1038/nm.4517.Our understanding of the link between the human microbiome and disease, including obesity, inflammatory bowel disease, arthritis and autism, is rapidly expanding. Improvements in the throughput and accuracy of DNA sequencing of the genomes of microbial communities associated with human samples, complemented by analysis of transcriptomes, proteomes, metabolomes and immunomes, and mechanistic experiments in model systems, have vastly improved our ability to understand the structure and function of the microbiome in both diseased and healthy states. However, many challenges remain. In this Review, we focus on studies in humans to describe these challenges, and propose strategies that leverage existing knowledge to move rapidly from correlation to causation, and ultimately to translation.Many of the studies described here in our laboratories were supported by the NIH, NSF, DOE, and the Alfred P. Sloan Foundation.2018-10-1

Molecular and pathological signatures of epithelial–mesenchymal transitions at the cancer invasion front

Reduction of epithelial cell–cell adhesion via the transcriptional repression of cadherins in combination with the acquisition of mesenchymal properties are key determinants of epithelial–mesenchymal transition (EMT). EMT is associated with early stages of carcinogenesis, cancer invasion and recurrence. Furthermore, the tumor stroma dictates EMT through intensive bidirectional communication. The pathological analysis of EMT signatures is critically, especially to determine the presence of cancer cells at the resection margins of a tumor. When diffusion barriers disappear, EMT markers may be detected in sera from cancer patients. The detection of EMT signatures is not only important for diagnosis but can also be exploited to enhance classical chemotherapy treatments. In conclusion, further detailed understanding of the contextual cues and molecular mediators that control EMT will be required in order to develop diagnostic tools and small molecule inhibitors with potential clinical implications

OPPOSING FUNCTIONS FOR A PROTEIN KINASE: A JNK1 DEPENDENT SWITCH DETERMINES THE ONCOGENIC OR TUMOR SUPPRESSIVE ACTIVITY OF ILK INRHABDOMYOSARCOMA

Background:The integrin-linked kinase (ILK) is a protein kinase involved in the regulation of pathogenic cancer cell behaviours, such as proliferation, survival and invasion. ILK appears to be pro-oncogenic in vitro and in vivo models of tumorigenesis. Rhabdomyosarcoma (RMS) is a primitive mesenchyme-derived tumor and is subclassified into primarily embryonal (ERMS) and alveolar (ARMS) variants. Patients who present with metastatic RMS tumors have a less than 20% chance of cure, suggesting a need to define novel targets for chemotherapeutic intervention. Methods: We used cell culture, murine xenografts and primary human tumors to examine ILK expression and functionality. RNAi and adenoviruses were used to knock down or over expressproteins, and SP600125 was used to inhibit JNK kinase activity. ERMS cells stablye xpressing PAX3-FOXO1A we regenerated using pcDNA3.1 with the full length PAX3-FOXO1A cDNA insert. Results: RNAi-mediated ablation of ILK induced stimulation of ERMS and inhibition of ARMS cell growth in vitro and in vivo. Overexpression of ILK, but not the ILK-R211A mutant reversed these effects. High-throughput screening of multiple tumor cell lines and mesenchymal progenitor cells demonstrated similar ILK anti-growth effects. Consistent with these results, clinical correlations made between ILK immunohistochemical staining intensity and patterns on an ERMS tumor tissue microarray revealed downregulation of ILK in stage III/IV primary tumors. Mechanistically, ILK silencing induced selective phosphorylation of the c-jun amino terminal kinase (JNK) and its target c-Jun in ERMS cells with attenuated phosphorylation in ARMS cells. ERMS cells express higher levels of JNK1 isoforms than ARMS cells. Introduction of the ARMS-associated PAX3-FOXO1A fusion gene into ERMS cells restored the oncogenic function of ILK and downregulated of JNK1. Coupling ILK siRNA with inhibition of the JNK-c-Jun signaling pathway in ERMS cells resulted in growth reductions and apoptotic induction. In contrast, coupling ILK knockdown with overexpression of JNK1 in ARMS cells resulted in growth and c-jun phosphorylation. Conclusion: In summary, these data suggest a model whereby the effect of ILK as an oncogene or tumor suppressor is determined by JNK1. Finally, this data suggests that ILK kinase inhibition may be warranted in ARMS tumors, and may be contraindicated in ERMS

Integrin-Linked Kinase Mediates Bone Morphogenetic Protein 7-Dependent Renal Epithelial Cell Morphogenesis

Bone morphogenetic protein 7 (BMP7) stimulates renal branching morphogenesis via p38 mitogen-activated protein kinase (p38(MAPK)) and activating transcription factor 2 (ATF-2) (M. C. Hu, D. Wasserman, S. Hartwig, and N. D. Rosenblum, J. Biol. Chem. 279:12051-12059, 2004). Here, we demonstrate a novel role for integrin-linked kinase (ILK) in mediating renal epithelial cell morphogenesis in embryonic kidney explants and identify p38(MAPK) as a target of ILK signaling in a cell culture model of renal epithelial morphogenesis. The spatial and temporal expression of ILK in embryonic mouse kidney cells suggested a role in branching morphogenesis. Adenovirus-mediated expression of ILK stimulated and expression of a dominant negative ILK mutant inhibited ureteric bud branching in embryonic mouse kidney explants. BMP7 increased ILK kinase activity in inner medullary collecting duct 3 (IMCD-3) cells, and adenovirus-mediated expression of ILK increased IMCD-3 cell morphogenesis in a three-dimensional culture model. In contrast, treatment with a small molecule ILK inhibitor or expression of a dominant negative-acting ILK (ILK(E359K)) inhibited epithelial cell morphogenesis. Further, expression of ILK(E359K) abrogated BMP7-dependent stimulation. To investigate the role of ILK in BMP7 signaling, we showed that ILK overexpression increased basal and BMP7-induced levels of phospho-p38(MAPK) and phospho-ATF-2. Consistent with its inhibitory effects on IMCD-3 cell morphogenesis, expression of ILK(E359K) blocked BMP7-dependent increases in phospho-p38(MAPK) and phospho-ATF-2. Inhibition of p38(MAPK) activity with the specific inhibitor, SB203580, failed to inhibit BMP7-dependent stimulation of ILK activity, suggesting that ILK functions upstream of p38(MAPK) during BMP7 signaling. We conclude that ILK functions in a BMP7/p38(MAPK)/ATF-2 signaling pathway and stimulates epithelial cell morphogenesis

JNK1 determines the oncogenic or tumor-suppressive activity of the integrin-linked kinase in human rhabdomyosarcoma

Although most reports describe the protein kinase integrin-linked kinase (ILK) as a proto-oncogene, occasional studies detail opposing functions in the regulation of normal and transformed cell proliferation, differentiation, and apoptosis. Here, we demonstrated that ILK functions as an oncogene in the highly aggressive pediatric sarcoma alveolar rhabdomyosarcoma (ARMS) and as a tumor suppressor in the related embryonal rhabdomyosarcoma (ERMS). These opposing functions hinge on signaling through a noncanonical ILK target, JNK1, to the proto-oncogene c-Jun. RNAi-mediated depletion of ILK induced activation of JNK and its target, c-Jun, resulting in growth of ERMS cells, whereas in ARMS cells, it led to loss of JNK/c-Jun signaling and suppression of growth both in vitro and in vivo. Ectopic expression of the fusion gene characteristic of ARMS (paired box 3–forkhead homolog in rhabdomyosarcoma [PAX3-FKHR]) in ERMS cells was sufficient to convert them to an ARMS signaling phenotype and render ILK activity oncogenic. Furthermore, restoration of JNK1 in ARMS reestablished a tumor-suppressive function for ILK. These findings indicate what we believe to be a novel effector pathway regulated by ILK, provide a mechanism for interconversion of oncogenic and tumor-suppressor functions of a single regulatory protein based on the genetic background of the tumor cells, and suggest a rationale for tailored therapy of rhabdomyosarcoma based on the different activities of ILK