Activation and regulation of a p38α MAPK by its downstream MAPKAP kinase through feedback phosphorylation and LLPS-driven condensate formation

MAP kinases (MAPKs) represent a class of evolutionarily conserved signaling molecules that respond to an array of extracellular stimuli and stresses. p38 group of MAPKs have been implicated in a multitude of signal transduction pathways known to be activated by dual-specific upstream MAPK kinases and also by autophosphorylation. They activate MAPK activated protein kinases (MAPKAPKs) in a context dependent manner by specific phosphorylation, and together they play crucial biological roles. One such pair in C. elegans consists of PMK3, p38α-MAPK and its cognate MAPKAPK, MAK2 downstream of DLK1 (MAPK kinase kinase) and MKK4 (MAPK kinase). They are implicated in axonal regeneration, degeneration and synaptic pruning in response to neuronal injury. Here, we report that PMK3 participates in a feedback loop with MAK2 leading to phosphorylation-mediated activation of both kinases. Interestingly, MAK2 that is a Ser/Thr kinase phosphorylates PMK3 at its TxY-motif required for the manifestation of full activity of PMK3. This observation identifies MAK2 as a dual specificity kinase. Distribution of phosphorylation sites on either kinase and presence of long intrinsically disordered regions in each of them indicate the possibility of conformational plasticity in the PMK3:MAK2 complex. Furthermore, MAK2 increases bioavailability of aggregation-prone PMK3 by forming LLPS-driven condensates. PMK3 and MAK2 retain the feedback phosphorylation relationship and enzymatic activities in the condensate state as well. Our observations establish an unreported feedback relationship between a MAPK and its downstream MAPKAPK through phosphorylation and LLPS-driven condensate formation.

Significance Statement

MAPK family of protein kinases are evolutionarily conserved and participate in a diverse array of signaling events. p38 MAPKs are key players in cellular phenomenon involving cell survival, differentiation, metabolic processes, and neuronal response to injury. They are activated through phosphorylation by upstream MAPK kinases (MAP2Ks) or by autophosphorylation. We found that a C. elegans MAPK (PMK3) engages in a feedback phosphorylation loop with its downstream effector kinase MAK2, but not the upstream MAP2K MKK4, that ensures robust activation of PMK3 by MAK2 without requiring MKK4. In addition, MAK2 increases bioavailability of activation-competent and active PMK3 by preventing its aggregation through LLPS-driven condensate formation. This feedback relationship between a MAPK and its downstream MAPKAPK might ensure rapid activation of this pathway in response to nervous system injury or stress.

Highlights

• PMK3 participates in a feedback phosphorylation loop with its downstream kinase MAK2 that leads to the phosphorylation of the activating TxY motif.

• MAK2 Phosphorylates PMK-3 at Ser, Thr and Tyr residues and that is a dual specificity kinase rather than a Ser/Thr kinase.

• MAK2 but not PMK3 alone forms condensates through liquid liquid phase separation (LLPS).

• While PMK3 alone is prone to aggregation, MAK2 prevents aggregation of PMK3 via LLPS-mediated condensate formation when present together.