Crystal structure of bisphosphorylated IGF-1 receptor kinase: insight into domain movements upon kinase activation.

BACKGROUND: The insulin-like growth-factor-1 (IGF-1) receptor, which is widely expressed in cells that have undergone oncogenic transformation, is emerging as a novel target in cancer therapy. IGF-1-induced receptor activation results in autophosphorylation of cytoplasmic kinase domains and enhances their capability to phosphorylate downstream substrates. Structures of the homologous insulin receptor kinase (IRK) exist in an open, unphosphorylated form and a closed, trisphosphorylated form. RESULTS: We have determined the 2.1 A crystal structure of the IGF-1 receptor protein tyrosine kinase domain phosphorylated at two tyrosine residues within the activation loop (IGF-1RK2P) and bound to an ATP analog. The ligand is not in a conformation compatible with phosphoryl transfer, and the activation loop is partially disordered. Compared to the homologous insulin receptor kinase, IGF-1RK2P is trapped in a half-closed, previously unobserved conformation. Observed domain movements can be dissected into two orthogonal rotational components. CONCLUSIONS: Conformational changes upon kinase activation are triggered by the degree of phosphorylation and are crucially dependent on the conformation of the proximal end of the kinase activation loop. This IGF-1RK structure will provide a molecular basis for the design of selective antioncogenic therapeutic agents.

MYCN oncogene and angiogenesis: down-regulation of endothelial growth inhibitors in human neuroblastoma cells. Purification, structural, and functional characterization.

Angiogenesis, the formation of new blood vessels, is seen during embryonic development and tumor progression, but the mechanisms have remained unclear. Recent data indicate that tumor angiogenesis can be induced by cellular oncogenes, leading to the enhanced activity of molecules stimulating angiogenesis. However, activated oncogenes might also facilitate angiogenesis by down-regulating endogenous inhibitors of angiogenesis. We report here that enhanced expression of the N-myc oncogene in human neuroblastoma cells down-regulates three inhibitors of endothelial cell proliferation. One of them was identified by amino acid sequencing as being identical with activin A, a developmentally-regulated protein. Down-regulation involves interaction of the N-myc protein with the activin A promoter. Work is ongoing to characterize the other two endothelial cell inhibitors. We suggest that the N-myc induced down-regulation of angiogenesis inhibitors could contribute to tumor angiogenesis.

Liposomes containing interferon-gamma as adjuvant in tumor cell vaccines.

PURPOSE: Liposomal systems may be useful as a cytokine supplement in tumor cell vaccines by providing a cytokine reservoir at the antigen presentation site. Here, we examined the effect of liposome incorporation of mIFNgamma on its potency as adjuvant in an established tumor cell vaccination protocol in the murine B16 melanoma model. Adjuvanticity of the mIFNgamma-liposomes was compared to that achieved by mIFNgamma-gene transfection of the B16 tumor cells. Furthermore, we studied whether liposomal incorporation of mIFNgamma indeed increases the residence time of the cytokine at the vaccination site. METHODS: C57B1/6 mice were immunized with i) irradiated IFNgamma-gene transfected B16 melanoma cells or ii) irradiated wild type B16 cells supplemented with (liposomal) mIFNgamma, followed by a challenge with viable B16 cells. The residence time of the (liposomal) cytokine at the subcutaneous (s.c.) vaccination site was monitored using radiolabeled mIFNgamma and liposomes. RESULTS: Immunization with irradiated tumor cells admixed with liposomal mIFNgamma generated comparable protection against B16 challenge as immunization with mIFNgamma-gene modified tumor cells. Irradiated tumor cells admixed with soluble mIFNgamma did not generate any protective responses. Radiolabeling studies indicated that free mIFNgamma rapidly cleared from the s.c. injection site. Association of [125I]-mIFNgamma with liposomes increased the local residence time substantially: liposomal association of mIFNgamma resulted in a prolonged local residence time of the cytokine as reflected by a 4-fold increase of the area under the curve. The amount of released cytokine in the optimal dose range corresponds to the amount released by the gene-transfected cells. Moderate but significant CTL-activity against B16 cells was found for mice immunized with irradiated cells supplemented with mIFNgamma-liposomes compared to untreated control animals. CONCLUSIONS: Prolonged presence of mIFNgamma at the site of antigen presentation is crucial for the generation of systemic immune responses in the B16 melanoma model. These studies show that liposomal encapsulation of cytokines is an attractive strategy for paracrine cytokine delivery in tumor vaccine development.

2A proteinases of coxsackie- and rhinovirus cleave peptides derived from eIF-4 gamma via a common recognition motif.

The cleavage specificities of the 2A proteinases from coxsackievirus B4 (CVB4) and human rhinovirus 2 (HRV2) on oligopeptide substrates have been determined. Comparison of the specificity of CVB4 2A proteinase with that of HRV2 2A proteinase allowed cleavable peptides to be designed using the common motif IIe/Leu-X-Thr-X*Gly; little resemblance to the viral cleavage site remained. The data also allowed the prediction of three possible cleavage sites for 2A proteinases on eIF-4 gamma; two peptides derived from these sequences were cleaved by both 2A proteinases. One of these peptides corresponds to the cleavage site for 2A proteinases mapped on eIF-4 gamma [B. J. Lamphear et al. (1993) J. Biol. Chem. 268, 19200-19203]. This supports the hypothesis that cleavage of eIF-4 gamma by picornaviral 2A proteinases occurs directly.