MAMMALIAN MITOGEN-ACTIVATED PROTEIN-KINASE PATHWAYS ARE REGULATED THROUGH FORMATION OF SPECIFIC KINASE-ACTIVATOR COMPLEXES

Mammalian cells contain at least three signaling systems which are str ucturally related to the mitogen-activated protein kinase (MAPK) pathw ay, Growth factors acting through Ras primarily stimulate the Raf/MEK/ MAPK cascade of protein kinases, In contrast, many stress-related sign als such as heat shock, inflammatory cytokines, and hyperosmolarity in duce the MEKK/SEK(MKK4)/SAPK(JNK) and/or the MKK3 or MKK6/p38(hog) pat hways. Physiological agonists of these pathway types are either qualit atively or quantitatively distinct, suggesting few common proximal sig naling elements, although past studies performed in vitro, or in cells using transient over-expression, reveal interaction between the compo nents of all three pathways, These studies suggest a high degree of cr oss-talk apparently not seen in vivo. We have examined the possible mo lecular basis of the differing agonist profiles of these three MAPK pa thways, We report preferential association between MAP kinases and the ir activators in eukaryotic cells. Furthermore, using the yeast 8-hybr id system, we show that association between these components can occur independent of additional eukaryotic proteins, We show that SAPK(JNK) or p38(hog) activation is specifically impaired by co-expression of c ognate dominant negative MAP kinase kinase mutants, demonstrating func tional specificity at this level. Further divergence and insulation of the stress pathways occurs proximal to the MAPK kinases since activat ion of the MAPK kinase kinase MEKK results in SAPK(JNK) activation but does not cause p38(hog) phosphorylation, Therefore, in intact cells, the three MAPK pathways may be independently regulated and their compo nents show specificity in their interaction with cognate cascade membe rs, The degree of intermolecular specificity suggests that mammalian M APK signaling pathways may remain distinct without the need for specif ic scaffolding proteins to sequester components of individual pathways .