SMAD proteins of oligodendroglial cells regulate transcription of JC virus early and late genes coordinately with the Tat protein of human immunodeficiency virus type 1.

JC virus (JCV) is the aetiological agent of progressive multifocal leukoencephalopathy (PML), a fatal, demyelinating disease of the brain affecting people with AIDS. Although immunosuppression is involved in infection of the brain by JCV, a direct influence of human immunodeficiency virus type 1 (HIV-1) has also been established. The Tat protein of HIV-1 has been implicated in activation of the cytokine transforming growth factor (TGF)-beta in HIV-1-infected cells and in stimulating JCV gene transcription and DNA replication in oligodendroglia, the primary central nervous system cell type infected by JCV in PML. This study demonstrated that Tat can cooperate with SMAD proteins, the intracellular effectors of TGF-beta, at the JCV DNA control region (CR) to stimulate JCV gene transcription. Tat stimulated JCV early gene transcription in KG-1 oligodendroglial cells when expressed via transfection or added exogenously. Using chromatin immunoprecipitation, it was shown that exogenous Tat enhanced binding of SMAD2, -3 and -4 and their binding partner Fast1 to the JCV CR in living cells. When SMAD2, -3 and -4 were expressed together, Tat, expressed from plasmid pTat, stimulated transcription from both early and late gene promoters, with the early promoter exhibiting stimulation of >100-fold. Tat, SMAD4 and JCV large T-antigen were all visualized in oligodendroglial cells at the border of an active PML lesion in the cerebral frontal lobe. These results revealed a positive reinforcement system in which the SMAD mediators of the TGF-beta system act cooperatively with Tat to stimulate JCV gene transcription.

New concepts regarding focal adhesion kinase promotion of cell migration and proliferation.

Focal adhesion kinase (FAK) is a non-receptor cytoplasmic tyrosine kinase that plays a key role in the regulation of proliferation and migration of normal and tumor cells. FAK associates with integrin receptors and recruits other molecules to the site of this interaction thus forming a signaling complex that transmits signals from the extracellular matrix to the cell cytoskeleton. Crk-associated substrate (CAS) family members appear to play a pivotal role in FAK regulation of cell migration. Cellular Src bound to FAK phosphorylates CAS proteins leading to the recruitment of a Crk family adaptor molecule and activation of a small GTPase and c-Jun N-terminal kinase (JNK) promoting membrane protrusion and cell migration. The relocalization of CAS and signaling through specific CAS family members appears to determine the outcome of this pathway. FAK also plays an important role in regulating cell cycle progression through transcriptional control of the cyclin D1 promoter by the Ets B and Kruppel-like factor 8 (KLF8) transcription factors. FAK regulation of cell cycle progression in tumor cells requires Erk activity, cyclin D1 transcription, and the cyclin-dependent kinase (cdk) inhibitor p27Kip1. The ability of FAK to integrate integrin and growth factor signals resulting in synergistic promotion of cell migration and proliferation, and its potential regulation by nuclear factor kappa B (NFkappaB) and p53 and a ubiquitously expressed inhibitory protein, suggest that it is remarkable in its capacity to integrate multiple extracellular and intracellular stimuli.

Lyn kinase activity is the predominant cellular SRC kinase activity in glioblastoma tumor cells.

Cellular Src activity modulates cell migration, proliferation, and differentiation, and recent reports suggest that individual members of the Src family may play specific roles in these processes. As we have found that Lyn, but not Fyn, activity promotes migration of glioblastoma cells in response to the cooperative signal generated by platelet-derived growth factor receptor beta and integrin alpha(v)beta3, we compared the activity and expression of Lyn and Fyn in glioblastoma (grade IV) tumor biopsy samples with that in anaplastic astrocytoma (grade III) tumors, nonneoplastic brain, and normal autopsy brain samples. Lyn kinase activity was significantly elevated in glioblastoma tumor samples. Notably, the Lyn kinase activity accounted for >90% of pan-Src kinase activity in glioblastoma samples but only approximately 30% of pan-Src kinase activity in the other groups. The levels of phosphorylation of the autophosphorylation site were consistent with significantly higher Lyn activity in glioblastoma tumor tissue than nonneoplastic brain. Although the normalized levels of Lyn protein and the relative levels of Lyn message were significantly higher in glioblastoma samples than nonneoplastic brain, the normalized levels of Lyn protein did not correlate with Lyn activity in the glioblastoma samples. There was no significant difference in the normalized levels of c-Src and Fyn protein and message in the glioblastoma and nonneoplastic brain. Immunostaining revealed that Lyn is located primarily in the glioblastoma cells in the tumor biopsies. These data indicate that Lyn kinase activity is significantly elevated in glioblastoma tumors and suggest that it is the Lyn activity that promotes the malignant phenotype in these tumors.