The interaction of p38 with its upstream kinase MKK6
Mitogen-activated protein kinase (MAPK; p38, ERK and JNK) cascades are evolutionarily conserved signaling pathways that regulate the cellular response to a variety of extracellular stimuli, such as growth factors and interleukins. The MAPK p38 is activated by its specific upstream MAPK kinases, MKK6 and MKK3. However, a comprehensive molecular understanding of how these cognate upstream kinases bind and activate p38 is still missing. Here, we combine NMR spectroscopy and isothermal titration calorimetry to define the binding interface between full-length MKK6 and p38. We show that p38 engages MKK6 not only via its hydrophobic docking groove, but also helix F, a secondary structural element that plays a key role in organizing the kinase core. We also show that, unlike MAPK phosphatases, the p38 conserved docking (CD) site is much less affected by MKK6 binding. Finally, we demonstrate that these interactions with p38 are conserved independent of the MKK6 activation state. Together, our results reveal differences between specificity markers of p38 regulation by upstream kinases, which do not effectively engage the CD site, and downstream phosphatases, which require the CD site for productive binding.