Current Biology 18, 311–321, March 11, 2008 ª2008 Elsevier Ltd All rights reservedDOI 10.1016/j.cub.2008.02.006
by MST1 and MST2
Inhibits Cell Proliferation
Maria Praskova,1,2,3Fan Xia,1,2,3and Joseph Avruch1,2,*
1Diabetes Unit and Medical Services and
Department of Molecular Biology
Massachusetts General Hospital
Boston, Massachusetts 02114
2Department of Medicine
Harvard Medical School
Boston, Massachusetts 02114
Background: MST1 and MST2 are the mammalian Ste20-
related protein kinases most closely related to Drosophila
Hippo, a major regulator of cell proliferation and survival
during development. Overexpression of MST1 or MST2 in
mammalian cells is proapototic; however, little is known
concerning the physiologic regulation of the endogenous
MST1/MST2 kinases,theirrole inmammaliancellproliferation,
or the identity of the MST1/MST2 substrates critical to prolifer-
Results: We show that MST1 and MST2 activity increases
where these kinases exhibit both an increase in both abun-
dance and activation. MST1 and MST2 also can be activated
nonphysiologically by okadaic acid or H2O2. The MOBKL1A
and MOBKL1B polypeptides, homologs of the Drosophila
MATS polypeptide, are identified as preferred MST1/MST2
substrates in vitro and are phosphorylated in cells in an
MST1/MST2-dependent manner in mitosis and in response
to okadaic acid or H2O2. MST1/MST2-catalyzed MOBKL1A/
MOBKL1B phosphorylation alters the ability of MOBKL1A/
MOBKL1B to bind and regulate downstream targets such as
the NDR-family protein kinases. Thus, MOBKL1A/MOBKL1B
phosphorylation in cells promotes MOBKL1A/MOBKL1B
binding to the LATS1 kinase and enables H2O2-stimulated
LATS1 activation loop phosphorylation. Most importantly,
replacement of endogenous MOBKL1A/MOBKL1B by a
nonphosphorylatable mutant is sufficient to accelerate cell
proliferation substantially by speeding progression through
G1/S as well as mitotic exit.
activated in mitosis and catalyze the mitotic phosphorylation
of MOBKL1A/MOBKL1B. MOBKL1A/MOBKL1B phosphoryla-
tion, in turn, is sufficient to inhibit proliferation through actions
at several points in the cell cycle.
identified by their Ste20-related catalytic domain  and
independently as KRS1 and KRS2, protein kinases activated
late after transformation by v-src . The regulation and
physiologic functions of these two kinases in mammalian cells
are not completely understood. Early studies showed that
overexpression of MST1 or MST2 in mammalian cells
promotes apoptosis; in addition, apoptosis, once initiated by
a variety of stimuli, results in the activation of MST1/MST2,
and a caspase-catalyzed cleavage generates a highly active
catalytic fragment [4–6]. MST1 also can be activated in cells
by okadaic acid, H2O2, or heat shock at 55?C. A more physio-
logic activation of MST1 is reflected by the requirement for
a Nore1B/RAPL-MST1 complex in the rap1-induced increase
inintegrin avidity thatoccursinresponsetoTCRorchemokine
stimulation of murine T cells , although the MST1 substrates
responsible for this action are as yet unknown.
MST1 and MST2 are the closest mammalian orthologs ofthe
Drosophila Hippo protein kinase (for reviews, see [8–10]), and
human MST2 (but not MST1) can complement Hippo
deficiency in the fly . Hippo deficiency in the developing
eye results in massive overgrowth due to an accelerated rate
of proliferation and a failure of developmental apoptosis. The
phenotype of Hippo deficiency is very similar to that seen
with loss of function (LOF) of Warts/LATS, another protein
kinase. Warts is orthologous to the murine LATS1 kinase,
and biallelic deletion of the murine LATS1 gene results in soft
tissue sarcomas and ovarian tumors . The noncatalytic
protein Salvador/Shar-pei, whose deficiency results in a phe-
notype resembling a weak LOF of both Hippo and Warts/
LATS, binds to both kinases, and is believed to facilitate
Hippo-catalyzed phosphorylation and activation of Warts/
LATS. In turn, Warts/LATS phosphorylates and inhibits the
phenocopies the LOF of Hippo and Warts/LATS . LOF of
the Drosophila MATS gene yields a phenotype closely resem-
bling those of Hippo and Warts/LATS ; MATS is homolo-
gous to the S. cerevisae polypeptides Mob1 and Mob2,
which act during mitotic exit to facilitate the ability of Cdc15,
a Hippo/MST1-2 homolog, to phosphorylate and activate
Wei et al. found that MATS is a Hippo substrate that serves
a similar role in the Hippo activation of Warts/LATS . In
turn, recent evidence indicates that mammalian MST2 can
phosphorylate and activate LATS1  and that the LATS1
and LATS2 kinases also may regulate mitotic exit [19, 20].
Other candidate MST substrates are histone H2B, which is
phosphorylated at Ser14 during apoptosis, possibly by a
caspase-cleaved catalytic fragment of MST1 ; and
the FoxO1 and 3 polypeptides, whose MST1-catalyzed
phosphorylation interferes with negative regulation of FoxO
by Akt .
Thus, kinases orthologous with MST1 and MST2 appear to
contribute to a variety of cellular programs, including mitotic
progression, integrin affinity, apoptosis, and tumor suppres-
sion. Herein we identify the MOBKL1A/MOBKL1B polypep-
tides as major physiologic substrates of MST1/MST2 and
MOBKL1A/MOBKL1B binding to NDR-family kinases. Impor-
tantly, we demonstrate that the MST1/MST2-catalyzed
phosphorylation of MOBKL1A/MOBKL1B in intact cells is
sufficient to substantially retard cell-cycle progression.
3These authors contributed equally to this work.
Screening for MST1 Substrates In Vitro
Extracts of murine L1210 preB-cell leukemia cells (ATCC
CCL219) were fractionated on anion and cation exchange
columns, and fractions were briefly phosphorylated in vitro
 with recombinant MST1, which had been preactivated in
vitro by incubation with Mg + ATP . The most prominent
MST1-phosphorylated band was a polypeptide of approxi-
mately 25 kDa recovered in the eluate of the cation exchange
column (Figure 1A). Gel filtration of the pooled fractions
containing this putative substrate resulted in a large portion
emerging just behind the void volume, Mr > 500 kDa, with
the remainder eluting at a molecular weight of approximately
30 kDa. The latter peak was pooled and subjected to
SDS-PAGE, and the polypeptides migrating between Mr
20–40 kDa were digested in situ with trypsin; the tryptic
peptides were analyzed by ESI-MS-MS. The largest number
of peptides identified corresponded to peroxyredoxin1;
however, a substantial number of peptides corresponded to
MOBKL1A and MOBKL1B, mammalian homologs of the yeast
Mob and Drosophila MATS polypeptides. The mouse and
human genomes each contain two genes encoding MATS-
related polypeptides that are identical between the two
species and that differ from each other at only 8/216 amino
acids (96.3% identity); the human polypeptide described by
NP_775739 is MOBKL1A (encoded on Chr 4q13.3), and the
human polypeptide described by NP_060691 is MOBKL1B
Figure 1. Identification and Characterization of a Substrate for the MST1 and MST2 Kinases
(A) Shown is the detection of an MST1 substrate in vitro. L1210 cell extract was fractionated by NaCl gradient elution. Fractions were subjected to
phosphorylation 6 MST1 as described in the Supplemental Experimental Procedures.
(B) Time course of MST1-catalyzed32P incorporation into MOBKL1A (-), histone H2B (:), and Peroxiredoxin-1 (Prx-1) (>).
(C) The effect of mutation of MOBKL1A Thr12 and Thr35 on MOBKL1A phosphorylation by MST1.
(D) Different MOB family members as substrates for MST1/MST2/MST3. Substrates were phosphorylated by preactivated MST1 (solid lines),MST2 (dashed
lines), and MST3 as in (B) for the time points indicated. The insert shows a Coomassie blue stain reflecting the relative amount of the substrates in the
reaction. The open symbols are MST1, and the closed symbols are MST2. The panel below the graph is an anti-FLAG blot reflecting the relative amount
of each kinase in the reaction. The autoradiograph shows the relative amount of32P incorporation into the kinases and the substrates.
Current Biology Vol 18 No 5
14. Lai, Z.C., Wei, X., Shimizu, T., Ramos, E., Rohrbaugh, M., Nikolaidis, N.,
Ho, L.L., and Li, Y. (2005). Control of cell proliferation and apoptosis by
mob as tumor suppressor, mats. Cell 120, 675–685.
15. Simanis, V. (2003). Events at the end of mitosis in the budding and
fission yeasts. J. Cell Sci. 116, 4263–4275.
16. Bardin, A.J., and Amon, A. (2001). Men and sin: what’s the difference?
Nat. Rev. Mol. Cell Biol. 2, 815–826.
17. Wei, X., Shimizu, T., and Lai, Z.C. (2007). Mob as tumor suppressor is
activated by Hippo kinase for growth inhibition in Drosophila. EMBO
J. 26, 1772–1781.
18. Chan, E.H., Nousiainen, M., Chalamalasetty, R.B., Scha ¨fer, A., Nigg,
E.A., and Sillje ´, H.H. (2005). The Ste20-like kinase Mst2 activates the
human large tumor suppressor kinase Lats1. Oncogene 24, 2076–2086.
19. Bothos, J., Tuttle, R.L., Ottey, M., Luca, F.C., and Halazonetis, T.D.
(2005). Human LATS1 is a mitotic exit network kinase. Cancer Res. 65,
20. Yabuta, N., Okada, N., Ito, A., Hosomi, T., Nishihara, S., Sasayama, Y.,
Fujimori, A., Okuzaki, D., Zhao, H., Ikawa, M., et al. (2007). Lats2 is an
essential mitotic regulator required for the coordination of cell division.
J. Biol. Chem. 282, 19259–19271.
21. Cheung, W.L., Cheung, W.L., Ajiro, K., Samejima, K., Kloc, M., Cheung,
P., Mizzen, C.A., Beeser, A., Etkin, L.D., Chernoff, J., et al. (2003).
Apoptotic phosphorylation of histone H2B is mediated by mammalian
sterile twenty kinase l. Cell 113, 507–517.
22. Lehtinen, M.K., Yuan, Z., Boag, P.R., Yang, Y., Villen, J., Becker, E.B.,
DiBacco, S., de la Iglesia, N., Gygi, S., Blackwell, T.K., and Bonni, A.
oxidative-stress responses and extends life span. Cell 125, 987–1001.
23. Knebel, A., Morrice, N., and Cohen, P. (2001). A novel method to identify
protein kinase substrates: eEF2 kinase is phosphorylated and inhibited
by SAPK4/p38delta. EMBO J. 20, 4360–4369.
24. Praskova, M., Khoklatchev, A., Ortiz-Vega, S., and Avruch, J. (2004).
Regulation of the MST1 kinase by autophosphorylation, by the growth
inhibitory proteins, RASSF1 and NORE1 and by Ras. Biochem. J. 381,
25. Hergovich, A., Stegert, M.R., Schmitz, D., and Hemmings, B.A. (2006).
NDR kinases regulate essential cell processes from yeast to humans.
Nat. Rev. Mol. Cell Biol. 7, 253–264.
26. Mah, A.S., Jang, J., and Deshaies, R.J. (2001). Protein kinase Cdc15
activates the Dbf2-Mob1 kinase complex. Proc. Natl. Acad. Sci. USA
27. Hou, M.C., Salek, J., and McCollum, D. (2000). Mob1p interacts with the
Sid2p kinase and is required for cytokinesis in fission yeast. Curr. Biol.
28. Hou, M.C., Wiley, D.J., Verde, F., and McCollum, D. (2003). Mob2p
interacts with the protein kinase Orb6p to promote coordination of
cell polarity with cell cycle progression. J. Cell Sci. 116, 125–135.
29. Bichsel, S.J., Tamaskovic, R., Stegert, M.R., and Hemmings, B.A.
(2004). Mechanism of activation of NDR (nuclear Dbf2-related) protein
kinase by the hMOB1 protein. J. Biol. Chem. 279, 35228–35235.
30. Luca, F.C., Mody, M., Kurischko, C., Roof, D.M., Giddings, T.H., and
Winey, M. (2001). Saccharomyces cerevisiae Mob1p is required for
cytokinesis and mitotic exit. Mol. Cell. Biol. 21, 6972–6983.
31. Hou, M.C., Guertin, D.A., and McCollum, D. (2004). Initiation of cytokine-
sis is controlled through multiple modes of regulation of the
Sid2p-Mob1p kinase complex. Mol. Cell. Biol. 24, 3262–3276.
32. Hergovich, A., Schmitz, D., and Hemmings, B.A. (2006). The human
tumour suppressor LATS1 is activated by human MOB1 at the
membrane. Biochem. Biophys. Res. Commun. 345, 50–58.
33. Stegert, M.R., Hergovich, A., Tamaskovic, R., Bichsel, S.J., and
Hemmings, B.A. (2005). Regulation of NDR protein kinase by hydropho-
bic motif phosphorylation mediated by the mammalian Ste20-like
kinase MST3. Mol. Cell. Biol. 25, 11019–11029.
34. Harvey, K.F., Pfleger, C.M., and Hariharan, I.K. (2003). The Drosophila
Mst ortholog, hippo, restricts growth and cell proliferation and
promotes apoptosis. Cell 114, 457–467.
MOBKL1A/MOBKL1B Phosphorylation Slows Proliferation