PURIFICATION AND IDENTIFICATION OF A MAJOR ACTIVATOR FOR P38 FROM OSMOTICALLY SHOCKED CELLS - ACTIVATION OF MITOGEN-ACTIVATED PROTEIN-KINASE-6 BY OSMOTIC SHOCK, TUMOR-NECROSIS-FACTOR-ALPHA, AND H2O2

A stress-activated, serine/threonine kinase, p38 (also known as HOG1 o r MPK2) belongs to a subgroup of mitogen-activated protein kinase (MAP K) superfamily molecules, An activity to activate p38 (p38 activator a ctivity) as well as p38 activity itself were greatly stimulated by hyp erosmolar media in mouse lymphoma L5178Y cells, The activator activity has been purified by sequential chromatography. A 36-kDa polypeptide that was coeluted with the activity in the final chromatography step w as identified as MAPK kinase 6 (MAPKK6) by protein microsequencing ana lysis, Monoclonal and polyclonal antibodies raised against recombinant MAPKK6 recognized specifically the 36-kDa MAPKK6 protein but did not cross react with MKK3 proteins. The use of these anti-MAPKK6 antibodie s revealed that two major peaks of the p38 activator activity in the f irst chromatography step reside in the activated MAPKK6, Using a genet ic screen in yeast, we isolated MKK3b, an alternatively spliced form o f MKK3. Like MKK3 and MAPKK6, MKK3b was shown to be a specific activat or for p38 and was activated by osmotic shock when expressed in COS7 c ells, Immunoblotting analysis revealed that MAPKK6 is expressed highly in HeLa and KB cells and scarcely in PC12 cells, whereas MKK3 and MKK 3b are expressed in all cells examined, Immunodepletion of MAPKK6 from the extracts obtained from L5178Y cells and KB cells exposed to hyper osmolar media depleted them of almost all of the p38 activator activit y, indicating that MAPKK6 is a major activator for p38 in an osmosensi ng pathway in these cells. In addition, MAPKK6 was activated strongly by tumor necrosis factor-alpha, H2O2, and okadaic acid and moderately by cycloheximide in KB cells. Thus, there are at least three members o f p38 activator, MKK3, MKK3b, and MAPKK6, and MAPKK6 may function as a major activator for p38 when expressed.