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

The cJun N-terminal kinases (JNKs) are encoded by three genes generating te n protein kinase polypeptides and are activated in settings of cell stress, mitogenesis, differentiation and morphogenesis. The specific role of the J NK family members in these diverse cell programmes is largely undefined. In this study, we tested the hypothesis that individual JNK isoforms would ex hibit distinct patterns of regulation within cells. The cDNAs encoding five haemagglutinin (HA)-tagged JNK isoforms (p46JNK1 alpha, p54JNK2 alpha, p54 JNK2 beta, p46JNK3 and p54JNK3) were expressed in cultured rat PCl2 phaeoch romocytoma cells and human small-cell lung cancer (SCLC) cells by retroviru s-mediated gene transfer. In addition, HA-tagged forms of the dual-specific ity mitogen-activated protein kinase kinases (MKKs), MKK4 and MKK7, which a re specific activators of the JNK enzymes, were similarly expressed. Revers e transcription and PCR revealed that JNK3 is endogenously expressed in SCL C cells, but not in either chromaffin or neuronally differentiated PCl2 cel ls. MKK4 and MKK7 were endogenously expressed in both PCl2 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. Hype rtonic stress, elicited by mannitol, also significantly stimulated these sa me JNKs, although the JNK3 isoforms were most strongly activated. In PCl2 c ell transfectants, however, selective and equal activation of p54JNK2a: and p54JNK3 by UV and osmotic stress was observed, with little or no activatio n of JNK1 alpha or JNK2 beta. 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.