Several protein kinases were found to interact with ABI1 and ABI2 in yeast two-hybrid screens. SOS2 (salt overly sensitive protein kinase, also known as Ca2+ sensor-interacting protein kinase, a member of the SNF1-related protein kinase SnRK3 group) and several SOS2-like protein kinases interact with ABI1 and ABI2 through a protein kinase interaction motif (PKI) identified within the catalytic part of the phosphatases. Interestingly, the abi2-1 mutation located in the PKI domain disrupts this interaction and at the same time enhances plant tolerance to salt stress (Ohta et al. 2003). ABI2 and to a lesser extent ABI1 interact with PKS3 (SOS2-like protein kinase 3), a negative regulator of the ABA pathway. The abi1-1 and abi2-1 mutants are able to suppress the pks3 ABA hypersensitive phenotype. Thereby, ABI2, ABI1 and PKS3 form a calcium-responsive negative regulatory loop together with the calcium binding protein CaBP5 in fine-tuning the plant sensitivity to ABA (Guo et al. 2002). Another ABA- and osmotic stress-activated protein kinase SnRK2E/OST1/SnRK2.6 (Mustilli et al. 2002; Yoshida et al. 2002) interacts with ABI1 possibly through PKI, as the abi1-1 mutation reduces this binding (Yoshida et al. 2006). Distinct roles of ABI1 and ABI2 were demonstrated by suppression of the ABA-dependent activation of SRK2E/OST1 that was observed in abi1-1 but not in abi2-1 mutant. Similar wilty phenotypes of srk2e/ost1 and abi1-1 plants suggest that ABI1 positively regulates the activation of this kinase (Yoshida et al. 2002, 2006). A further ABI1-interacting protein is the ABA- and drought-inducible transcription factor ATHB6. This interaction depends on the catalytic activity of the phosphatase. ATHB6 negatively regulates ABA signalling. ABI1 acts upstream of ATHB6, as ABA induction of the ATHB6 promoter-reporter is abolished in abi1-1 plants (Himmelbach et al. 2002). The interaction of ABI2 and, to a lesser extent, of ABI1 with AtGPX3, a glutathione peroxidase, provides a link between ABA and H2O2 signalling in stomatal closure. AtGPX3 is involved in ABA-mediated production of H2O2, which affects the redox states of ABI1, ABI2 and AtGPX3 (Miao et al. 2006). H2O2 reversible inactivation of ABI1 and ABI2 proteins and their susceptibility to phenylarsine oxide already suggested the oxidation of critical cysteine residue(s) in the phosphatase (Meinhard and Grill 2001; Meinhard et al. 2002). The ability of oxidized AtGPX3 to reduce the phosphatase activi ties of ABI1 and ABI2 and enhanced insensitivity of the atgpx3 abi2-1 double mutants to ABA suggested that AtGPX3 may modulate the activities of ABI2 under oxidative stress in plants (Miao et al. 2006).
ABI1 interacts with phosphatidic acid (PA), which is produced in response to ABA application through phospholipase Da1. PA binding decreases the PP2C activity and affects its membrane association. The Arg-73 is essential for the ABI1-PA interaction (Zhang et al. 2004). ABI1-R73A mutant plants are insensitive to ABA-induced stomatal closure, emphasizing the requirement for PA binding to ABI1 in this process, but not in the ABA-induced inhibition of stomatal opening, which is regulated by interaction of PA and PLDa1 with the Ga subunit of heterotrimeric G protein (Mishra et al. 2006). Involvement of other PP2Cs in the regulation of ABA pathway is indicated by the ABA-induced PP2C activity measurement, where ABI1 and ABI2 contributed by approx. 50% to total PP2C-related activity (Merlot et al. 2001) and is further supported by additional studies of other A-type PP2C members.
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