Osmostress induces autophosphorylation of Hog1 via a C-terminal regulatory region that is conserved in p38α.

Many protein kinases require phosphorylation at their activation loop for induction of catalysis. Mitogen-activated protein kinases (MAPKs) are activated by a unique mode of phosphorylation, on neighboring Tyrosine and Threonine residues. Whereas many kinases obtain their activation via autophosphorylation, MAPKs are usually phosphorylated by specific, dedicated, MAPK kinases (MAP2Ks). Here we show however, that the yeast MAPK Hog1, known to be activated by the MAP2K Pbs2, is activated in pbs2Δ cells via an autophosphorylation activity that is induced by osmotic pressure. We mapped a novel domain at the Hog1 C-terminal region that inhibits this activity. Removal of this domain provides a Hog1 protein that is partially independent of MAP2K, namely, partially rescues osmostress sensitivity of pbs2Δ cells. We further mapped a short domain (7 amino acid residues long) that is critical for induction of autophosphorylation. Its removal abolishes autophosphorylation, but maintains Pbs2-mediated phosphorylation. This 7 amino acids stretch is conserved in the human p38α. Similar to the case of Hog1, it's removal from p38α abolishes p38α's autophosphorylation capability, but maintains, although reduces, its activation by MKK6. This study joins a few recent reports to suggest that, like many protein kinases, MAPKs are also regulated via induced autoactivation.

When expressed in yeast, mammalian mitogen-activated protein kinases lose proper regulation and become spontaneously phosphorylated.

MAPKs (mitogen-activated protein kinases) are key components in cell signalling pathways. Under optimal growth conditions, their activity is kept off, but in response to stimulation it is dramatically evoked. Because of the high degree of evolutionary conservation at the levels of sequence and mode of activation, MAPKs are believed to share similar regulatory mechanisms in all eukaryotes and to be functionally substitutable between them. To assess the reliability of this notion, we systematically analysed the activity, regulation and phenotypic effects of mammalian MAPKs in yeast. Unexpectedly, all mammalian MAPKs tested were spontaneously phosphorylated in yeast. JNKs (c-Jun N-terminal kinases) lost their phosphorylation in pbs2Delta cells, but p38s and ERKs (extracellular-signal-regulated kinases) maintained their spontaneous phosphorylation even in pbs2Deltaste7Deltamkk1Deltamkk2Delta cells. Kinase-dead variants of ERKs and p38s were phosphorylated in strains lacking a single MEK (MAPK/ERK kinase), but not in pbs2Deltaste7Deltamkk1Deltamkk2Delta cells. Thus, in yeast, p38 and ERKs are phosphorylated via a combined mechanism of autophosphorylation and MEK-mediated phosphorylation (any MEK). We further addressed the mechanism allowing mammalian MAPKs to exploit yeast MEKs in the absence of any activating signal. We suggest that mammalian MAPKs lost during evolution a C-terminal region that exists in some yeast MAPKs. Indeed, removal of this region from Hog1 and Mpk1 rendered them spontaneously and highly phosphorylated. It implies that MAPKs possess an efficient inherent autoposphorylation capability that is suppressed in yeast MAPKs via a C-terminal domain and in mammalian MAPKs via as yet unknown means.

JX401, A p38alpha inhibitor containing a 4-benzylpiperidine motif, identified via a novel screening system in yeast.

In vivo screening of compounds for potential pharmacological activity is more advantageous than in vitro screening. In vivo screens eliminate the isolation of compounds that cannot cross biological membranes, are cytotoxic, or are not specific to the target. However, animal-based or even cell-based systems are usually expensive, time-consuming, and laborious. Here we describe the identification of inhibitors of the mitogen-activated protein kinase p38alpha via a high throughput screen using yeast cells. p38alpha is hyperactive in inflammatory diseases, and various indications suggest that its inhibition would reverse inflammation. However, there are currently no p38alpha inhibitors in clinical use. Because the human p38alpha imposes severe growth retardation when expressed in yeast, we screened a library of 40,000 randomly selected small molecules for compounds that would restore a normal growth rate. We identified two compounds; both share a structural motif of 4-benzylpiperidine, and both were shown to be efficient and selective p38alpha inhibitors in vitro. They were also active in mammalian cells, as manifested by their ability to reversibly inhibit myoblast differentiation. Thus, the yeast screen identified efficient and specific p38alpha inhibitors that are capable of crossing biological membranes, are not toxic, and function in mammalian cells. The rapid and cost-efficient high-throughput screening used here could be applied for isolation of inhibitors of various targets.