Nutrient and growth factor receptors internalize through dathrin coated pits. The signal sequences which mediate the association between receptors and the coated pit reside in receptor cytoplasmic tail domains. These signal sequences have been extensively investigated in nutrient receptors, and a minimal functional sequence has been identified consisting of a tyrosine residue in an exposed b turn. Protein-protein contacts between internalization signal sequences and components of the coated pit machinery have been proposed to mediate rapid internalization. In vitro evidence suggests the AP-2 adaptor may be that protein component. The signal sequences of growth factor receptors are less well understood. However, a growth factor- and temperature- dependent binding between the epideimal growth factor receptor and the AP-2 adaptor has been observed. We identified Hsc70 as a cytosolic ligand for the cytoplasmic tail of the transferrin receptor. The binding was mapped to the internalization signal sequence of the receptor tail. Mutations within the signal sequence which inhibit internalization result in alteration of signal sequence secondary structure and reduction in stimulation of the Hsc70 ATPase. Co-immunoprecipitation analysis showed a population of transferrin receptors which are bound to Hsc70, suggesting an association in vivo. We also showed binding of Hsc70 to the epidermal growth factor receptor by co-immunoprecipitation analysis. This binding was increased by treatment with EGF. The binding was transient, and occured prior to the binding of the receptor to AP-2 adaptors. Other agents which induce EGF receptor clustering and internalization also stimulate the transient increase in Hsc70 binding and the later AP-2 binding, suggesting a role in early endocytosis. These data support the hypothesis that Hsc70 is associated with the receptors for transferrin and epidermal growth factor in vitro and in vivo. We propose a role for the 70 kD heat shock protein in the assembly/disassembly of protein complexes involved in receptor signalling and/or internalization.

The c-Jun NH2-terminal kinases (JNK) are evolutionarily conserved serine/threonine protein kinases that are activated by proinflammatory cytokines, environmental stress, and genotoxic agents. These kinases play key regulatory roles within a cell by coordinating signals from the cell surface to nuclear transcription factors. JNK phosphorylates the amino terminal domain of all three Jun transcription factors (JunB, c-Jun and JunD) all members of the AP-1 family. The activated transcription factors modulate gene expression to generate appropriate biological responses, including cell migration, proliferation, differentiation and cell death. The role of the JNK signaling pathway in cell death/apoptosis is controversial, both pro-apoptotic and pro-survival roles have been attributed to JNK. The mechanism that enables the JNK signaling pathway to mediate both apoptosis and survival is unclear. The aim of this study is to examine the role of TNF-stimulated JNK activation on cell survival. The proinflammatory cytokine TNF, is known to activate JNK and induce apoptosis. To test whether the JNK signaling pathway contributes to TNF-induced apoptosis, the response of wild type and Jnk1-/- Jnk2-/- (JNK deficient fibroblasts) fibroblasts to TNF was examined. JNK deficient fibroblasts are more sensitive to TNF-induced apoptosis than wild-type fibroblasts. The TNF-sensitivity cannot be attributed to altered expression of TNF receptors or defects in the NF-кB or AKT pathways, known anti-apoptotic signal transduction pathways. (In fact, TNF stimulated NF-кB activation provides a major mechanism to account for survival in both wild-type and JNK deficient cells.) However this increased TNF-sensitivity can be attributed to JNK deficiency. Apoptosis is suppressed in JNK deficient cells when transduced with JNK1 retrovirus. These data implicate the JNK signaling pathway in cell survival. The AP-1 family of transcription factors is a target of the JNK signal transduction pathway. In addition JNK is required for the normal expression of the AP-1 family member, JunD. Previous studies have indicated that JunD can mediate survival. Interestingly, JNK deficient and JunD null cells display similar phenotypes: premature senescence and increased sensitivity to TNF induced apoptosis. In fact, the TNF-sensitivity is also suppressed in JNK deficient fibroblasts transduced with JunD retrovirus. Although JunD can replace the survival signaling role of JNK, phosphorylation of JunD is essential to inhibit TNF induced apoptosis. JNK deficient cells transduced with phosphomutant JunD retrovirus maintain TNF-sensitivity. Activated transcription factors modulate gene expression. It is most likely that JunD functions by regulating the expression of key molecules that act to inhibit TNF-stimulated apoptosis. Microarray analysis comparing wild-type with JNK deficient fibroblasts revealed that the expression of the survival gene, cIAP-2, was induced by TNF in only wild-type fibroblasts. Furthermore, protein expression of cIAP-2 was induced by TNF in only wild-type fibroblasts. Analysis of the cIAP-2 promoter revealed two critical NF-кB binding sites and one AP-1 binding site. Luciferase reporter assays indicated key roles for both NF-кB and the AP-1 component, JunD in TNF-induced cIAP-2 gene expression. These experiments establish that the JNK/JunD pathway collaborates with NF-кB pathway to increase the expression of the anti-apoptotic protein cIAP-2 in TNF treated cells. Without this collaboration, the JNK pathway mediates apoptosis. The integration of JNK signaling with other signaling pathways represents a mechanism to account for the dual ability of the JNK pathway to mediate either survival or apoptosis. The dynamic coordination of signals within and between pathways is critical. The future challenge will be to fit the details of individual signaling pathways into the context of signaling networks.