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Chemokine receptors control several fundamental cellular processes in both hematopoietic and structural cells, including directed cell movement, i.e., chemotaxis, cell differentiation, and proliferation. We have previously demonstrated that CXCR3, the chemokine receptor expressed by Th1/Tc1 inflammatory cells present in the lung, is also expressed by human airway epithelial cells. In airway epithelial cells, activation of CXCR3 induces airway epithelial cell movement and proliferation, processes that underlie lung repair. The present study examined the expression and function of CXCR3 in human alveolar type II pneumocytes, whose destruction causes emphysema. CXCR3 was present in human fetal and adult type II pneumocytes as assessed by immunocytochemistry, immunohistochemistry, and Western blotting. CXCR3-A and -B splice variant mRNA was present constitutively in cultured type II ceils, but levels of CXCR3-B greatly exceeded CXCR3-A mRNA. In cultured type II cells, I-TAC, IP-10, and Mig induced chemotaxis. Overexpression of CXCR3-A in the A549 pneumocyte cell line produced robust chemotactic responses to I-TAC and IP-10. In contrast, I-TAC did not induce chemotactic responses in CXCR3-B and mock-transfected cells. Finally, I-TAC increased cytosolic [Ca.sup.2+] and activated the extracellular signal-regulated kinase, p38, and phosphatidylinositol 3-kinase (PI 3-kinase)/protein kinase B kinases only in CXCR3-A-transfected cells. These data indicate that the CXCR3 receptor is expressed by human type II pneumocytes, and the CXCR3-A splice variant mediates chemotactic responses possibly through [Ca.sup.2+] activation of both mitogen-activated protein kinase and PI 3-kinase signaling pathways. Expression of CXCR3 in alveolar epithelial cells may be important in pneumocyte repair from injury. lung inflammation; lung repair; chronic obstructive pulmonary disease

The p38 mitogen-activated protein kinase (MAPK) pathway plays an important role in cell differentiation, but the signaling mechanisms by which it is activated during this process are largely unknown. Cdo is an immuno-globulin superfamily member that functions as a component of multiprotein cell surface complexes to promote myogenesis. In this study, we report that the Cdo intracellular region interacts with JLP, a scaffold protein for the p38[alpha]/[beta] MAPK pathway. Cdo, JLP, and p38[alpha]/[beta] form complexes in differentiating myoblasts, and Cdo and JLP cooperate to enhance levels of active p38[alpha]/[beta] in transfectants. Primary myoblasts from [Cdo.sup.-/-] mice, which display a defective differentiation program, are deficient in p38[alpha]/[beta] activity, and the expression of an activated form of MKK6 (an immediate upstream activator of p38) rescues the ability of [Cdo.sup.-/-] cells to differentiate. These results document a novel mechanism of signaling during cell differentiation: the interaction of a MAPK scaffold protein with a cell surface receptor.

The JNK-interacting leucer zipper protein (JLP) which is a scaffolding protein involved in linking JNK signaling module components and G(alpha)(sub 13) proteins which are the alpha subunits of the heterometric G protein are studied. Results indicate that G(alpha)(sub 13) physically interacts with JLP and the presence of a ternary complex consisting of G(alpha)(sub 13), JLP and JNK indicates that JLP is a novel scaffolding protein in the G(alpha)(sub 13) mediated JNK signaling pathway.

Chemokine receptors control several fundamental cellular processes in both hematopoietic and structural cells, including directed cell movement, i.e., chemotaxis, cell differentiation, and proliferation. We have previously demonstrated that CXCR3, the chemokine receptor expressed by Th1/Tc1 inflammatory cells present in the lung, is also expressed by human airway epithelial cells. In airway epithelial cells, activation of CXCR3 induces airway epithelial cell movement and proliferation, processes that underlie lung repair. The present study examined the expression and function of CXCR3 in human alveolar type II pneumocytes, whose destruction causes emphysema. CXCR3 was present in human fetal and adult type II pneumocytes as assessed by immunocytochemistry, immunohistochemistry, and Western blotting. CXCR3-A and -B splice variant mRNA was present constitutively in cultured type II cells, but levels of CXCR3-B greatly exceeded CXCR3-A mRNA. In cultured type II cells, I-TAC, IP-10, and Mig induced chemotaxis. Overexpression of CXCR3-A in the A549 pneumocyte cell line produced robust chemotactic responses to 1-TAC and IP-10. In contrast, 1-TAC did not induce chemotactic responses in CXCR3-B and mock-transfected cells. Finally, I-TAC increased cytosolic Ca2+ and activated the extracellular signal-regulated kinase, p38, and phosphatidylinositol 3-kinase (PI 3-kinase)/protein kinase B kinases only in CXCR3-A-transfected cells. These data indicate that the CXCR3 receptor is expressed by human type II pneumocytes, and the CXCR3-A splice variant mediates chemotactic responses possibly through Ca2+ activation of both mitogen-activated protein kinase and PI 3-kinase signaling pathways. Expression of CXCR3 in alveolar epithelial cells may be important in pneumocyte repair from injury. [ABSTRACT FROM AUTHOR]

Scaffolding proteins play a critical role in conferring specificity and fidelity to signaling pathways. The JNK-interacting leucine zipper protein (JLP) has been identified as a scaffolding protein involved in linking components of the JNK signaling module. Gα12 and Gα13, the α-subunits of heterotrimteric G proteins G12 and G13, respectively, stimulate the JNK module in diverse cell types. Here, we report that Gα13 physically interacts with JLP, and this interaction enhances Gα13-mediated JNK activation. We also demonstrate endogenous interaction between JLP and Gα13 in MCF-7 cells. JLP interaction is specific to the G12 family of α-subunits via its C-terminal domain (termed GID-JLP), spanning amino acids 1165–1307, and this interaction is more pronounced with the mutationally or functionally activated form of Gα13 compared to that of wild-type Gα13. The presence of a ternary complex consisting of Gα13, JLP, and JNK suggests a role for JLP in tethering Gα13 to the signaling components involved in JNK activation. Coexpression of GID-JLP disrupts ternary complex formation in addition to attenuating Gα13-stimulated JNK activity. These findings identify JLP as a novel scaffolding protein in the Gα13- mediated JNK signaling pathway. [ABSTRACT FROM AUTHOR]

Scaffolding proteins exist in eukaryotes to properly assemble signaling proteins into specific multimeric functional complexes. JLP is a novel leucine zipper protein belonging to a family of scaffolding proteins that assemble JNK signaling modules. JLP is a proline-rich protein that contains two leucine zipper domains and a highly conserved C-terminal domain. We have identified kinesin light chain 1 (KLC1) as a binding partner for the second leucine zipper domain of JLP using yeast two-hybrid screening. The interaction domain of KLC1 was mapped to its tetratripeptide repeat, which contains a novel leucine zipper-like domain that is crucial for the interaction with JLP. Mutations of Leu-280, Leu-287, Val-294, and Leu-301 within this domain of KLC1 disrupted its ability to associate with JLP. Immunofluorescence studies showed that JLP and KLC1 co-localized in the cytoplasm and that the localization of JLP was dependent on its second leucine zipper. Ectopic expression of a dominant negative form of KLC1 resulted in the mislocalization of endogenous JLP. Moreover, the association between JLP and KLC1 occurred in vivo and was important in the formation of ternary complex with JNK1. These results identify a novel protein-protein interaction between KLC1 and JLP that involves leucine zipper-like domains and support the role of motor proteins in the spatial regulation of signaling modules. [ABSTRACT FROM AUTHOR]

JLP (JNK-associated leucine zipper protein) is a novel scaffolding protein involved in JNK signaling. Although it is known that JLP is highly expressed in brain, the biological function of JLP in neuronal systems remains unknown. Here, we report a novel interaction between JLP and SCG10 (superior cervical ganglia clone 10), which is a microtubule-destabilizing factor that is essential for neurite outgrowth. Inhibition of endogenous JLP expression using small interference RNA methodology strongly enhanced nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. Our results show that JLP negatively regulates NGF-induced neurite outgrowth by decreasing the level of phosphorylated SCG10. Furthermore, inhibition of JNK phosphorylation by a small molecule inhibitor, SP600125, resulted in inhibition of SCG 10 phosphorylation and inhibition of neurite growth. Taken together, our results suggest that JLP negatively regulates NGF-induced neurite outgrowth through a sequestering mechanism that results in an attenuation of NGF-induced SCG10 phosphorylation. [ABSTRACT FROM AUTHOR]