IDENTIFICATION OF ANISOMYCIN-ACTIVATED KINASES P45 AND P55 IN MURINE CELLS AS MAPKAP KINASE-2

We recently showed that EGF and anisomycin activate two kinases, p45 a nd p55, whose distinguishing feature is that their detection in in-gel kinase assays is enhanced by copolymerised poly-Glu/Tyr or poly-Glu/P he (Cane E, Hazzalin CA and Mahadevan LC, Mol. Cell, Biol,, 20:117-121 ). Their activation characteristics and sizes are strikingly similar t o those of JNK/SAPKs, which are also strongly activated by anisomycin. However, we show here that p45 and p55 are not JNK/SAPKs but murine f orms of MAPKAP kinase-2 because: (i) Detection of immunoprecipitated J NK/SAPKs is completely dependent on the presence of c-Jun as substrate in the in-gel kinase assays, whereas detection of p45 and p55 is not, (ii) Detection of p45 and p55 activity is enhanced by the presence of poly-Glu/Tyr or poly-Glu/Phe, whereas JNK/SAPKs are not detectable un der these conditions. (iii) Although the sizes of the murine JNK/SAPKs and MAPKAP K-2 are similar, human JNK/SAPKs migrate at 45 and 55 kDa whereas human MAPKAP K-2 migrates at 50 kDa; the poly-Glu/Tyr-enhanced activity in human cells migrates at 50 kDa. (iv) Purified rabbit musc le MAPKAP K-2 is detectable as two bands of activity on in-gel kinase assays and their detection is enhanced by poly-Glu/Tyr. (v) Finally, t he anisomycin-activated poly-Glu/Tyr-enhanced p45 and p55 kinases can be immunoprecipitated from murine cells using an anti-MAPKAP K-2 antib ody. Thus, EGF- and anisomycin-activated p45 and p55 are not JNK/SAPKs but MAPKAP K-2, implying that both these agents activate the p38/RK M AP kinase cascade.