Dose rate and mode of exposure are key factors in JNK activation by UV irradiation.

Single exposure of cells to UVC (254 nm for 30 s) or to UVB (300 nm for 10 min) was shown to activate jun-NH2 kinases which, in turn, phosphorylate their substrates ELK-1, c-jun and ATF-2. While UVC (40-80 J/m2) activates JNK up to 4 h, with maximal induction after 30 min, UVB (150-300 J/m2) activates JNK over a prolonged period, up to 24 h, with maximal induction after 6 h. UV-mediated activation of src-related tyrosine kinases and MAPK revealed different kinetics, with maximal induction after 24 h. As recent studies had indicated a role of a UVC component in mediating the ability of UVB to activate JNK, we have examined the effect of dose rate as well as of multiplicity of exposures on the activation of these kinases. The UVC portion found in 300 J/m2 UVB (5%, corresponding to 15 J/m2, administered within 10 s) did not activate JNK. However, when the same dose was administered at a lower rate (i.e. over 10 min, as needed for UVB irradiation) it was found capable of activating JNK, MAPK and src kinases, but to a lower degree and with different kinetics than found for UVB. Such differences point to cellular changes which are elicited by UVB, but not UVC. Although a single UVB exposure using a filter that blocks wavelengths below 300 nm prevented activation of JNK, multiple exposures of filtered UVB wavelengths (mimicking chronic exposure) were able to activate JNK. We conclude that the mode of UVB exposure (dose rate and multiplicity) is a crucial determinant for physiologically relevant activation of JNK.

Activation of c-Jun-NH2-kinase by UV irradiation is dependent on p21ras.

We have demonstrated previously that Jun-NH2-kinase (JNK) activation in vitro is potentiated by association with the p21(ras) protein. To determine if in vivo activation of JNK also depends on p21(ras), we have used M1311 cells that carry the cDNA for the neutralizing antibody to p21(ras), Y13-259, under a dexamethasone-inducible promoter. The ability of UV to activate JNK gradually decreased over a 4-day period of cell growth in dexamethasone. This decrease coincides with weaker transcriptional activation measured via gel shift and chloramphenicol acetyltransferase assays. Peptides corresponding to amino acids 96-110 on p21(ras), which were shown to block Ras-JNK association, inhibited UV-mediated JNK activation in mouse fibroblast 3T3-4A cells as well as in M1311 cells, further supporting the role of p21(ras) in UV-mediated JNK activation. Overall, the present studies provide in vivo confirmation of the role p21(ras) plays in JNK activation by UV irradiation.

Expression of a 32 kDa protein in rat mammary tumors induced by anti-benzo[c]phenanthrene-3,4-diol-1,2-epoxide.

Racemic anti-benzo[c]phenanthrene-3,4-diol-1,2-epoxide (BcPDE) is a powerful rat mammary carcinogen and a metabolite of benzo[c]phenanthrene, a polynuclear aromatic hydrocarbon found in the environment. In elucidating potential molecular mechanisms that may play a role in the development of BcPDE-induced rat mammary tumors, we have identified a 32 kDa protein in 16 of 26 tumors analyzed but in only 1 of the 15 normal mammary tissues that were examined. The 32 kDa protein was identified with antibodies to Ets, which also recognized the 55 kDa Ets-1 protein that was expressed at similar levels in normal mammary tissues. The expression of the 32 kDa protein was also observed in mammary tumor-derived cell lines of both rat and human origin and in human melanoma, but not in normal human keratinocytes or rat fibroblast cell lines. Further characterization via 2D gels revealed that the protein exhibits a PI of 5.5. Southwestern analysis using Ets-1 target sequence revealed binding of the 55 kDa Ets-1 but not of the newly identified 32 kDa protein. Overall, the preferential expression of the 32 kDa protein in mammary tumor tissues may serve as a biomarker to follow the development of this tumor type.

Mapping the intracytoplasmic regions of the alpha granulocyte-macrophage colony-stimulating factor receptor necessary for cell growth regulation.

The granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor is composed of an alpha subunit which binds GM-CSF and a beta subunit, which together form the high affinity receptor. By transfecting the human alpha subunit into murine Ba/F3 cells, we have been able to investigate the role of the short 54-amino acid intracytoplasmic portion (amino acid 346-400) of this subunit in mediating cell growth. We have shown that the intracytoplasmic amino acids 346-382 are necessary for GM-CSF-mediated cell growth. In contrast, amino acids 382-400 can be removed without effect. The stable transfection of the human beta subunit into the cell lines containing the mutant alpha subunits did not affect the growth characteristics of these cells. The ability of GM-CSF to stimulate cell growth of the Ba/F3 cells alpha subunit transfectants was correlated with the ability of this hormone to translocate protein kinase C to the particulate fraction. In contrast, the ability of GM-CSF addition to increase phosphorylation of the human beta subunit did not correlate with cell growth and required the entire intracytoplasmic domain of the alpha subunit. These results demonstrate an important role for the intracytoplasmic portion of the alpha subunit in mediating both signal transduction and cell cycle commitment stimulated by GM-CSF.

A critical role for the cytoplasmic domain of the granulocyte-macrophage colony-stimulating factor alpha receptor in mediating cell growth.

The human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor is composed of an alpha subunit which binds GM-CSF and a beta subunit which allows for high affinity binding. To investigate the role of the short cytoplasmic tail (54 amino acids) of the alpha receptor in mediating signal transduction and in controlling cell growth, we placed a stop codon after the alpha receptor transmembrane domain and expressed this receptor in murine Ba/F3 cells. Unlike the complete alpha subunit, this shortened receptor was unable to stimulate protein phosphorylation or mediate entry into the cell cycle. By comparing Ba/F3 cells expressing the alpha and beta receptors with those expressing the alpha or the terminated alpha receptor, we have been able to correlate specific GM-CSF-induced events with cell cycle commitment. We find that cell growth is correlated with prolonged increases in the cell levels of c-myc, pim-1, and cyclin D2 mRNAs, but not with changes in either immediate early genes or mitogen-activated protein kinase phosphorylation. This suggests that additional signal transduction pathways not mediated by known phosphoproteins are activated by GM-CSF. Since the beta receptor is shared by human interleukins 3 and 5, our data suggest that the specificity of response to GM-CSF is mediated in part by the short cytoplasmic tail of the alpha receptor subunit.

Affinity-purified c-Jun amino-terminal protein kinase requires serine/threonine phosphorylation for activity.

The addition of phorbol esters to U937 leukemic cells stimulates the phosphorylation of c-Jun on serines 63 and 73. To isolate the protein kinase which stimulates this phosphorylation, we have used heparin-Sepharose chromatography followed by affinity chromatography over glutathione-Sepharose beads bound with a fusion protein of glutathione S-transferase and amino acids 5-89 of c-Jun (GST-c-Jun). Using this procedure we purify a 67-kDa protein which is capable of phosphorylating GST-c-Jun as well as the complete c-Jun protein. By making mutations in serines 63 and 73 and then creating a fusion protein with GST (GST-c-Jun mut), we demonstrate that this protein kinase specifically phosphorylates these sites in the c-Jun amino terminus. Treatment of purified c-Jun amino-terminal protein kinase (cJAT-PK) with phosphatase 2A inhibits its ability to phosphorylate GST-c-Jun. This inactivated enzyme can be reactivated by phosphorylation with protein kinase C (PKC), although PKC is not capable of phosphorylating the GST-c-Jun substrate. Because v-Jun cannot be phosphorylated in vivo, we compared the ability of cJAT-PK to bind to GST-v-Jun or GST-c-Jun mut. The cJAT-PK bound 50-fold better to GST-c-Jun mut than GST-v-Jun suggesting that the delta domain which is missing in v-Jun plays a role in binding the cJAT-PK. These results suggest that there is a protein kinase cascade mediated by protein phosphatases and PKC which regulates c-Jun phosphorylation.

Long terminal repeats of dwarf hamster endogenous retrovirus are highly diverged and do not maintain efficient transcription.

Recently we described a new endogenous proretrovirus of dwarf hamster Phodopus sungorus (MRS-Ps). Its sequence possesses evident homology with the endonuclease domain of the mouse mammary tumor virus pol gene. Here we present nucleotide sequence data on three clones of retroviral long terminal repeats. As many as 15% of substituted, deleted, and inserted base pairs were found while comparing these sequences. Hence, MRS-Ps seems to be rather an old genetic element which originated about 30 million years ago. One LTR is 877 bp long and contains numerous elements that control its transcriptional activity: TATA-box, glucocorticoid responsive element, NF1-binding site, etc. Nevertheless, this LTR does not govern efficient transcription of adjacent genes in a transient expression assay. In addition, we failed to find MRS-specific mRNA in adult, embryonic, and mammary tumor cells.