The p38 mitogen-activated protein kinases are activated in response to various 
extracellular signals in eukaryotic cells and play a critical role in the 
cellular responses to these signals. The four mammalian isoforms (p38alpha, 
p38beta, p38gamma, and p38delta) are coexpressed and coactivated in the same 
cells. The exact role of each p38 isoform has not been entirely identified, in 
part due to the inability to activate each member individually. This could be 
resolved by the use of intrinsically active mutants. Based on previous studies 
on yeast p38/Hog1 [Bell M, Capone R, Pashtan I, Levitzki A & Engelberg D (2001) 
J Biol Chem276, 25351-2538] and human p38alpha[Diskin R, Askari N, Capone R, 
Engelberg D & Livnah O (2004) J Biol Chem279, 47040-47049] we have generated 
intrinsically active p38beta, p38gamma and p38delta mutants. In addition, we 
have identified a new activating mutation site in p38alpha. Most of the 
activating mutations are located in the L16 loop, in which conformational 
changes were shown to induce activation. We show that these changes impose 
substantial autophosphorylation activity, providing a mechanistic explanation 
for the intrinsic activity of the mutants. The new active variants maintain 
specificity towards substrates and inhibitors similar to that of the parental 
wild-type proteins, and are phosphorylated by mitogen-activated protein kinase 
kinase 6, their upstream activator. Thus, we have completed the development of a 
series of intrinsically active mutants of all p38 isoforms. These active 
variants could now become powerful tools for the elucidating the activation 
mechanism and specific biological roles of each p38 isoform.