Differential Modulation of the Phosphoproteome by the MAP Kinases Isoforms p38 and p38

The p38 members of the mitogen-activated protein kinases (MAPKs) family mediate various cellular responses to stress conditions, inflammatory signals, and differentiation factors. They are constitutively active in chronic inflammatory diseases and some cancers. The differences between their transient effects in response to signals and the chronic effect in diseases are not known. The family is composed of four isoforms, of which p38 & alpha; seems to be abnormally activated in diseases. p38 & alpha; and p38 & beta; are almost identical in sequence, structure, and biochemical and pharmacological properties, and the specific unique effects of each of them, if any, have not yet been revealed. This study aimed to reveal the specific effects induced by p38 & alpha; and p38 & beta;, both when transiently activated in response to stress and when chronically active. This was achieved via large-scale proteomics and phosphoproteomics analyses using stable isotope labeling of two experimental systems: one, mouse embryonic fibroblasts (MEFs) deficient in each of these p38 kinases and harboring either an empty vector or vectors expressing p38 & alpha;(WT), p38 & beta;(WT), or intrinsically active variants of these MAPKs; second, induction of transient stress by exposure of MEFs, p38 & alpha;(-/-), and p38 & beta;(-/-) MEFs to anisomycin. Significant differences in the repertoire of the proteome and phosphoproteome between cells expressing active p38 & alpha; and p38 & beta; suggest distinct roles for each kinase. Interestingly, in both cases, the constitutive activation induced adaptations of the cells to the chronic activity so that known substrates of p38 were downregulated. Within the dramatic effect of p38s on the proteome and phosphoproteome, some interesting affected phosphorylation sites were those found in cancer-associated p53 and Hspb1 (HSP27) proteins and in cytoskeleton-associated proteins. Among these, was the stronger direct phosphorylation by p38 & alpha; of p53-Ser309, which was validated on the Ser315 in human p53. In summary, this study sheds new light on the differences between chronic and transient p38 & alpha; and p38 & beta; signaling and on the specific targets of these two kinases.