DEF Pocket in p38α Facilitates Substrate Selectivity and Mediates Autophosphorylation

Netanel Tzarum,Nadav Komornik,Dorin Ben Chetrit,David Engelberg,Oded Livnah

doi:10.1074/jbc.m113.464511

Netanel Tzarum, Nadav Komornik + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m113.464511

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Abstract

Signaling processes are primarily promoted by molecular recognition and corresponding protein-protein interactions. One of the key eukaryotic signaling pathways is the MAP kinase cascade involved in vital cellular processes such as cell proliferation, differentiation, apoptosis, and stress response. The principle recognition site of MAP kinases, the common docking (CD) region, forms selective interactions with substrates, upstream activators, and phosphatases. A second docking site, defined as the DEF site interaction pocket (DEF pocket), is formed subsequent to ERK2 and p38α activation. Both crystal structures of p38α in its dually phosphorylated form and of intrinsically active mutants showed the DEF pocket, giving motivation for studying its role in substrate activation and selectivity. Mutating selected DEF pocket residues significantly decreased the phosphorylation levels of three p38α substrates (ATFII, Elk-1, and MBP) with no apparent effect on the phosphorylation of MK2 kinase. Conversely, mutating the CD region gave the opposite effect, suggesting p38α substrates can be classified into DEF-dependent and DEF-independent substrates. In addition, mutating DEF pocket residues decreased the autophosphorylation capability of intrinsically active p38α mutants, suggesting DEF-mediated trans-autophosphorylation in p38α. These results could contribute to understanding substrate selectivity of p38α and serve as a platform for designing p38α-selective DEF site blockers, which partially inhibit p38α binding DEF-dependent substrates, whereas maintaining its other functions intact. In this context, preliminary results using synthetic peptides reveal significant inhibition of substrate phosphorylation by activated p38α.

Highlights

Controlling vital cellular processes such as proliferation, differentiation, apoptosis, and stress response are primarily mediated by protein kinases
The principle docking site identified in all MAP kinases is defined as a common docking (CD)2 region that selectively interacts with corresponding substrates, upstream activators, and phosphatases
Catalytic studies of the p38␣D316N mutant revealed a similar kinetic profile to that of p38␣wt (Table 2). These results indicated that substrates that are less influenced by mutation in the CD domain are more dependent on interactions with the DEF pocket and vice versa

Summary

EXPERIMENTAL PROCEDURES

Site-directed Mutagenesis of the p38␣ Mutants and Structural Analysis—Site-directed mutagenesis was performed by polymerase chain reaction according to the recommendations of the manufacturer. Cells were collected by centrifugation and washed in phosphate-buffered saline (PBS) and the pellet was stored at Ϫ20 °C. A quality assay was done in which samples from the paper-spotted kinase reactions were mixed with Laemmli sample buffer and boiling at 100 °C for 5 min. The assay samples were run on SDS-PAGE stained with Coomassie staining and expose to x-ray film. The autophosphorylation kinase assay was performed in a similar buffer as the paper-spotted kinase assay with no substrate where each reaction contained 1.25 ␮g of purified protein in a final volume of 25 ␮l. The assay samples were run on SDS-PAGE with Coomassie staining and exposed to x-ray film. Cell Culture and Luciferase Assay—The transfected recombinant p38␣ cDNAs containing an HA tag were cloned into pCEFL vectors (Invitrogen). The cells were harvested 48 h post-transfection and the luciferase activity was measured using the dual luciferase reporter assay system (Promega)

RESULTS

Bond length Bond angle

DISCUSSION