Skip to main content
. Author manuscript; available in PMC: 2015 Dec 17.
Published in final edited form as: Curr Biol. 2008 Mar 11;18(5):311–321. doi: 10.1016/j.cub.2008.02.006

Figure 3. The Role of MST1/MST2 in MOBKL1A/MOBKL1B and LATS1 Phosphorylation in Response to Okadaic Acid and H2O2.

Figure 3

(A) Overexpression of a kinase-dead MST2 inhibits okadaic-acid-induced MOBKL1A/MOBKL1B and LATS1/LATS2 (Thr1079), but not LATS1/LATS2 (Ser909) phosphorylation. U2OS cells stably expressing a tetracycline (Tet) inducible Flag-MST2 (K56R) were treated with doxycycline (lanes 3 and 4) or carrier (lanes 1 and 2) and 12 hr later with okadaic acid (1 μM) (lanes 2 and 4) or carrier (lanes 1 and 3) for 30 min prior to harvest.

(B) Overexpression of a kinase-dead MST2 inhibits H2O2-induced MOBKL1A/MOBKL1B and phosphorylation of both LATS1/LATS2 (Thr1079) and LATS1 (Ser909). The experiment was carried out exactly as in (A), except the stimulation was with H2O2 (2.5 mM), which was added for 20 min.

(C) Replacement of endogenous MOBKL1A/MOBKL1B with recombinant MOBKL1A wild-type does not alter H2O2 stimulated LATS1/LATS2 phosphorylation. U2OS cells carry cassettes of shRNA against 3′ UTR of MOBKL1A, shRNA directed against the coding region of MOBKL1B, and cDNA encoding Flag-MOBKL1A. The first cassette is constitutively expressed, whereas the latter two are tetracycline inducible. Five days prior to stimulation, doxycycline (5 μg/ml) (lanes 3 and 4) or carrier (lanes 1 and 2) was added. Cells were treated with H2O2 as in (B) (lanes 2 and 4). Note that the recombinant Flag-MOBKL1A runs slightly slower than the endogenous MOBKLIA/MOBKL1B.

(D) Elimination of endogenous MOBKL1A/MOBKL1B phosphorylation selectively eliminates H2O2-stimulated phosphorylation of LATS1/LATS2 (Ser909), but not of LATS1/LATS2 (Thr1079). The experiment was carried out exactly as in (C), except that the cDNA sequences employed encodes MOBKL1A (Thr12/35Ala).