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The cJun N-terminal kinases (JNKs) are encoded by three genes generating ten protein kinase polypeptides and are activated in settings of cell stress, mitogenesis, differentiation and morphogenesis. The specific role of the JNK family members in these diverse cell programmes is largely undefined. In this study, we tested the hypothesis that individual JNK isoforms would exhibit distinct patterns of regulation within cells. The cDNAs encoding five haemagglutinin (HA)-tagged JNK isoforms (p46JNK1alpha, p54JNK2alpha, p54JNK2beta, p46JNK3 and p54JNK3) were expressed in cultured rat PC12 phaeochromocytoma cells and human small-cell lung cancer (SCLC) cells by retrovirus-mediated gene transfer. In addition, HA-tagged forms of the dual-specificity mitogen-activated protein kinase kinases (MKKs), MKK4 and MKK7, which are specific activators of the JNK enzymes, were similarly expressed. Reverse transcription and PCR revealed that JNK3 is endogenously expressed in SCLC cells, but not in either chromaffin or neuronally differentiated PC12 cells. MKK4 and MKK7 were endogenously expressed in both PC12 cells and SHP77 cells. Immunoprecipitation and analysis of the JNKs expressed in SCLC cells revealed strong stimulation of all five JNK isoforms by UV radiation. Hypertonic stress, elicited by mannitol, also significantly stimulated these same JNKs, although the JNK3 isoforms were most strongly activated. In PC12 cell transfectants, however, selective and equal activation of p54JNK2alpha and p54JNK3 by UV and osmotic stress was observed, with little or no activation of JNK1alpha or JNK2beta. In contrast with the broad activation of the JNK enzymes by UV in SCLC cells, only HA-MKK4 was stimulated by UV exposure in these cells, whereas osmotic stress stimulated both HA-MKK4 and HA-MKK7. These findings indicate selective activation of JNK and MKK isoforms in a manner that is dependent upon the specific cell stress and the cell type.

Stress- and cell type-dependent regulation of transfected c-Jun N-terminal kinase and mitogen-activated protein kinase kinase isoforms