Identification of novel phosphorylation sites in MSK1 by precursor ion scanning MS

MSK1 (mitogen- and stress-activated kinase 1) is a dual kinase domain protein that acts downstream of the ERK1/2 (extracellular-signal-regulated kinase 1/2) and p38 MAPK (mitogen-activated protein kinase) signalling pathways in cells. MSK1, and its related isoform MSK2, phosphorylate the transcription factors CREB (cAMP-response-element-binding protein) and ATF1 (activating transcription factor 1), and the chromatin proteins histone H3 and HMGN1 (high-mobility-group nucleosomal-binding protein 1) in response to either mitogenic stimulation or cellular stress. MSK1 activity is tightly regulated in cells, and activation requires the phosphorylation of MSK1 by either ERK1/2 or p38α. This results in activation of the C-terminal kinase domain, which then phosphorylates further sites in MSK1, leading to the activation of the N-terminal kinase domain and phosphorylation of substrates. Here, we use precursor ion scanning MS to identify five previously unknown sites in MSK1: Thr⁶³⁰, Ser⁶⁴⁷, Ser⁶⁵⁷, Ser⁶⁹⁵ and Thr⁷⁰⁰. One of these sites, Thr⁷⁰⁰, was found to be a third site in MSK1 phosphorylated by the upstream kinases ERK1/2 and p38α. Mutation of Thr⁷⁰⁰ resulted in an increased basal activity of MSK1, but this could be further increased by stimulation with PMA or UV-C radiation. Surprisingly, however, mutation of Thr⁷⁰⁰ resulted in a dramatic loss of Thr⁵⁸¹ phosphorylation, a site essential for activity. Mutation of Thr⁷⁰⁰ and Thr⁵⁸¹ to an alanine residue resulted in an inactive kinase, while mutation of both sites to an aspartic acid residue resulted in a kinase with a significant basal activity that could not be further stimulated. Together these results are consistent with a mechanism by which Thr⁷⁰⁰ phosphorylation relieves the inhibition of MSK1 by a C-terminal autoinhibitory helix and helps induce a conformational shift that protects Thr⁵⁸¹ from dephosphorylation.