Biochemical analysis of MST1 kinase: elucidation of a C-terminal regulatory region

Abstract

The MST1 kinase phosphorylates FoxO transcription factors in the cytosol and histone H2B in the nucleus to promote cellular apoptosis. In addition to a N-terminal kinase domain, MST1 contains C-terminal regulatory and dimerization regions that are cleaved upon nuclear transport. In this report, we investigate the role of the MST1 regulatory region and dimerization domain in MST1 activity toward FoxO and histone H2B substrates. We find that the MST1 regulatory region enhances FoxO phosphorylation while inhibiting histone H2B phosphorylation, consistent with the cellular properties of MST1. We also identify autophosphorylation sites within the MST1 regulatory region and show that both regulatory region phosphorylation and MST1 dimerization contribute to FoxO phosphorylation. Together, our studies provide new insights into how MST1 substrate selectivity is modulated with implications for understanding apoptotic signaling through MST1 kinase.

Figure 1

MST1 preparation and characterization. (A) The construct designs for the various protein fragments of MST1 used for this study are depicted. The various domains are abbreviated as follows: KD, kinase domain; RR, regulatory region; and DD, dimerization domain. (B) SDS page gel of the various recombinant protein constructs used in this study. The abbreviations are as follows: MST1-FL, full-length MST1; MST1-KD/RR, MST1 kinase with the regulatory region, spanning residues 1–380; and MST1-FL (TM), MST1-FL triple mutant with residues S340, T346, and T348 mutated to alanine. (C) Autoradiography gel showing the activity of MST1 toward phosphorylation of its substrates, FoxO1 and histone H2B. The signal depicted is the incorporation of the γ-³²P label onto FoxO and histone H2B. The autophosphorylation signal of MST1 is also visible as indicated. (D) Autoradiograph showing the effect of DNA binding on the phosphorylation activity of FoxO1 by MST1-FL in the presence and absence of its cognate DNA, the Daf-16 binding element

Figure 2

Kinetic analysis of MST1 protein constructs. (A) Lineweaver–Burk plot comparing the activity of MST1-FL and the kinase domain (MST1-KD) toward FoxO1. (B) Lineweaver–Burk plot depicting the activity of MST-KD toward histone H2B. (C) Lineweaver–Burk plot depicting the activity of MST1-FL toward histone H2B. (D) Summary of the various steady state parameters for the plots shown in panels A–C. (E and F) Autoradiography gel showing the activity of MST1-FL and MST1-KD toward FoxO1 and histone H2B, respectively. The gel shows the incorporation of the γ-³²P label as a function of substrate concentration. All reactions were carried out for 15 min and then stopped with the addition of SDS–PAGE loading buffer.

Figure 3

Autophosphorylation of MST1. (A) Autoradiogarphy comparing the autophosphorylation activity of equimolar amounts of MST1-FL and MST1-KD. (B) Autoradiogarphy showing the autophosphorylation activity of various GST-tagged MST1 fragments. MST-FL was used as the enzyme to monitor γ-³²P incorporation. (C) Autoradiogarphy and SDS page gel of the GST-tagged phosphorylated region (residues 338–354) of wild-type MST1 and the TM mutant. (D) Lineweaver–Burk plot depicting the phosphorylation of MST1-FL toward a MST1 C-terminal region spanning residues 338–487 that encompasses the RR and DD. (E) Lineweaver–Burk plot depicting N-terminal KD domain phosphorylation of MST1. A kinase dead version of the enzyme harboring a K59R mutation was used as a substrate. (F) Summary of the various steady state parameters for plots shown in panels E and F.

Figure 4

Effect of MST1 dimerization on kinase activity. (A) Sedimentation equilibrium data for MST1-FL were fitted with data from nine curves (three protein concentrations at three centrifugation speeds). A representative run at a centrifugation speed of 20000 rpm and protein concentration of 0.3 mg/mL is shown. The plots represent a best fit to a single-dimeric species model to which all nine curves were fitted. The bottom panel shows the experimental data (O) with the calculated fits (–). The top panel depicts the residuals of the fits. (B) Sedimentation equilibrium data for the dimerization deletion construct spanning residues 1–380 of MST1. The experiments was carried out as described for panel A under similar conditions. The plot represents a single-monomeric species model to which all the nine curves were fit. (C) Lineweaver–Burk plot comparing the activity of various MST1 constructs toward FoxO1. Kinetics was performed on both the dimerization deletion mutant (MST1-KD/RR, residues 1–380) and the phosphorylation defective triple mutant MST1 (TM). (D) Summary of the various steady state parameters for the plots shown in panel C.