Figure 2. Characterization of MOBKL1A/MOBKL1B Binding to MST1/MST2 and LATS1.
(A) MOBKL1B binds native MST2 in preference to kinase-dead MST2 or native MST1. HA-MOBKL1B was cotrasfected in HEK293T cells with the Flag-tagged MST1 and MST2 variants indicated.
(B) MST2 autophosphorylation enhances, whereas MST2-catalyzed MOBKL1A phosphorylation inhibits, the MST-MOBKL1A interaction in vitro. Immobilized GST (lanes 4, 7, 11, and 14), GST-MOBKL1A wild-type (lanes 3, 6, 10, and 13), and the GST-MOBKL1A (Thr12/35Ala) (lanes 2, 5, 9, and 12) were incubated with purified MST2, nonpreactivated (lanes 2–4 and 9–11), or preactivated (lanes 5–7 and 12–14), with (lanes 9–14) or without (lanes 2–7) ATP. Note that phosphorylation of MOBKL1A (lanes 9 and 12) reduces its reactivity with the anti-MOBKL1A/MOBKL1B antibody (lowest panel).
(C) Characterization of LATS1 binding to unphosphorylated and phosphorylated MOBKL1A in vitro is shown. Immobilized GST, GST-MOB1A wild-type, or GST-MOBKL1A (Thr12/35Ala) were preincubated with preactivated MST2, with (lanes 7–12) or without (lanes 1–6) MgCl2 and ATP. After washing, okadaic-acid-treated (lanes 2, 4, 6, 8, 10, and 12) or untreated (1, 3, 5, 7, 9, and 11) HEK293T cell lysates containing Flag-LATS1 were added for pull-down assay.
(D) Characterization of the effect of okadaic acid on the ability of recombinant MOBKL1A wild-type or MOBKL1A (Thr12/35Ala) to bind endogenous MST1/MST2 and LATS1 during transient expression is shown. HEK293T cells expressing FLAG-tagged MOBKL1A wild-type (lanes 1 and 2) or MOBKL1A (Thr12/T35Ala) (lanes 3 and 4) were treated with okadaic acid (OA) (lanes 2 and 4) or not (lanes 1 and 3). Aliquots of the cell extracts containing 2 mg protein were incubated with either okadaic acid (lanes 2 and 4) or alkaline phosphatase (lanes 1 and 3) at room temperature for 30 min. and then subjected to anti-Flag immunoprecipitation.