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

Abstract

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 (mitogenactivated 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 nucleosomalbinding 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: Thr 630 , Ser 647 , Ser 657 , Ser 695 and Thr 700 . One of these sites, Thr 700 , was found to be a third site in MSK1 phosphorylated by the upstream kinases ERK1/2 and p38α. Mutation of Thr 700 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 Thr 700 resulted in a dramatic loss of Thr 581 phosphorylation, a site essential for activity. Mutation of Thr 700 and Thr 581 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 Thr 700 phosphorylation relieves the inhibition of MSK1 by a C-terminal autoinhibitory helix and helps induce a conformational shift that protects Thr 581 from dephosphorylation.

MSK1 Regulates Homeostatic and Experience-Dependent Synaptic Plasticity

“…While there were no changes in protein levels of the related RSK2, levels of MSK1 protein were somewhat reduced in hippocampus and cerebellum, which may reflect some instability in the mutated protein. Indeed, we have previously observed reduced expression of MSK1 protein in mutants affecting MSK1 kinase activity (McCoy et al, 2005(McCoy et al, , 2007. Accordingly, at this stage, we cannot attribute the synaptic and experience-dependent phenotype of the MSK1 KD mutant exclusively to a loss of kinase activity.…”

Casein Kinase 2 interacts with human mitogen- and stress-activated protein kinase MSK1 and phosphorylates it at Multiple sites

“…The molecular mechanism of MSK1 activation is compelx. It requires the phosphorylation of MSK1 at Ser360 and Thr581 by either ERK1/2 or p38α (2)(3)(4)6). These events activate the C-terminal kinase domain of MSK1, which then autophosphorylates at Ser212, Ser376, and Ser381, resulting in the activation of the N-terminal kinase domain.…”

“…Then Ser750, Ser752 and Ser758 were phosphorylated by the N-terminal kinase domain (7). In addition to these eight phosphorylation sites, five novel sites, Thr630, Ser647, Ser657, Ser695 and Thr700 have been recently identified (6). Among these five sites, Thr700 was found to be the third site phosphorylated by ERK1/2 or p38α, perhaps playing a key role in the activation of MSK1.…”

MSK regulate TCR‐induced CREB phosphorylation but not immediate early gene transcription

“…The mitogen-and stress-activated kinases (MSK) contain two kinase domains, an N-terminal domain related to the AGC kinase family and a C-terminal domain related to the calmodulin kinase (CaMK) family [4]. MSK are activated by phosphorylation on at least three sites by the upstream MAPK [5,6]. This results in activation of the C-terminal domain, leading to autophosphorylation of MSK and activation of the N-terminal domain, which is then able to phosphorylate MSK substrates.…”