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evidence that the group-A PP2Cs directly interact with SnRK2s and inactivates
and dephosphorylates SnRK2s. In response to ABA, SnRK2s are activated in asso-
ciation with the internal phosphorylation of multiple serine/threonine residues
(involving Ser175) in its kinase activation loop. The same sites are dephosphoryl-
ated by PP2Cs, resulting in the inactivation of SnRK2s (Umezawa et al. 2009 ). In
addition, Vlad et al. ( 2009 , 2010 ), using a protein phosphatase profiling strategy
to screen for putative substrates of HAB1 PP2C, showed that SnRK2.6/OST1 is
one of the substrates of a HAB1 and identified Ser175 in SRK2.6/OST1 as a target
site of the PP2C that directly binds in vivo to the regulatory C-terminal domain of
SRK2.6/OST1. Further results revealed that ABI1, ABI2, and their mutant forms
display very similar substrate preferences as HAB1 and its mutant form hab1 G246D
(Vlad et al. 2009 , 2010 ). These data provide convincing evidence that SnRK2s are
direct targets of the group-A PP2Cs and lead to a model that PYR/PYL/RCAR
inhibits the PP2C-dependent negative regulation of SnRK2s in an ABA-modulated
fashion (Umezawa et al. 2009 ). In support of this model, Umezawa et al. ( 2009 )
reconstituted these three components in vitro using recombinant ABI1 or abi1 - 1 ,
PYR1, and GFP-tagged SnRK2s from the Arabidopsis cells. After incubation with
PYR1, PP2Cs, and SnRK2s in the presence or absence of ABA, SnRK2 activ-
ity was monitored via an in-gel phosphorylation assay, which showed that PYR1
inhibits ABI1-mediated inactivation of SnRK2 in an ABA-dependent manner;
however, a dominant mutation form of ABI1, abi1 - 1, inactivates SnRK2s even in
the presence of PYR1 and ABA, consistent with previous data that the dominant
mutation forms of ABI1 and ABI2, namely abi1 - 1 and abi2 - 1, lack RCAR/PYR-
binding ability (Ma et al. 2009 ; Park et al. 2009 ). These results also provide a
clear explanation for why abi1 - 1 and abi2 - 1 are dominantly insensitive to ABA
(Umezawa et al. 2009 ; see also Chap. 8 ) . This model was further supported by
reconstituting in vitro the PYR/PYL/RCAR-PP2C-SnRK2.6-ABF2-linked signal-
ing cascades in the Arabidopsis protoplasts, in which ABA-triggered phosphoryla-
tion and activation of the transcription factor depends on the introduction of these
four components into plant protoplasts (Fujii et al. 2009 ). It was testified again
that, in the presence of ABA, the PYR/PYL/RCAR receptor proteins (with the
exception of PYL13) can disrupt the interaction between the PP2Cs and SnRK2s,
thus preventing the PP2C-mediated dephosphorylation of the SnRK2s and result-
ing in the activation of the SnRK2 kinases (Fujii et al. 2009 ).
Most recently, the Arabidopsis glycogen synthase kinase 3 (GSK3)-like kinases
(BIN2 and BIN2-like BIL1/2) that regulate negatively brassinosteroid signaling
(He et al. 2002 ; Li and Nam 2002 ) were shown to be positive regulators of ABA
signaling (Cai et al. 2014 ). BIN2 kinase interacts with, phosphorylates and acti-
vates SnRK2.2 and SnRK2.3 kinases to function in ABA signaling downstream
of PYR1/PYL/RCAR receptors and PP2Cs (Cai et al. 2014 ), suggesting a regu-
lation mechanism of SnRK2s by a reversible phosphorylation process where the
BIN2/BIL kinase-catalyzed phosphorylation cooperates with the PP2C-mediated
dephosphorylation of SnRK2s in regulating the SnRK activities.
The ABA-responsive bZIP transcription factors (ABFs) were shown to be
substrates of SnRK2s, which interact with, are phosphorylate by, and function
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