Arabidopsis ABA-Activated Kinase MAPKKK18 is Regulated by Protein Phosphatase 2C ABI1 and the Ubiquitin–Proteasome Pathway

Abstract

Phosphorylation and dephosphorylation events play an important role in the transmission of the ABA signal. Although SnRK2 [sucrose non-fermenting1-related kinase2] protein kinases and group A protein phosphatase type 2C (PP2C)-type phosphatases constitute the core ABA pathway, mitogen-activated protein kinase (MAPK) pathways are also involved in plant response to ABA. However, little is known about the interplay between MAPKs and PP2Cs or SnRK2 in the regulation of ABA pathways. In this study, an effort was made to elucidate the role of MAP kinase kinase kinase18 (MKKK18) in relation to ABA signaling and response. The MKKK18 knockout lines showed more vigorous root growth, decreased abaxial stomatal index and increased stomatal aperture under normal growth conditions, compared with the control wild-type Columbia line. In addition to transcriptional regulation of the MKKK18 promoter by ABA, we demonstrated using in vitro and in vivo kinase assays that the kinase activity of MKKK18 was regulated by ABA. Analysis of the cellular localization of MKKK18 showed that the active kinase was targeted specifically to the nucleus. Notably, we identified abscisic acid insensitive 1 (ABI1) PP2C as a MKKK18-interacting protein, and demonstrated that ABI1 inhibited its activity. Using a cell-free degradation assay, we also established that MKKK18 was unstable and was degraded by the proteasome pathway. The rate of MKKK18 degradation was delayed in the ABI1 knockout line. Overall, we provide evidence that ABI1 regulates the activity and promotes proteasomal degradation of MKKK18.

AIK1, A Mitogen-Activated Protein Kinase, Modulates Abscisic Acid Responses through the MKK5-MPK6 Kinase Cascade

“…3C). Similarly, ABI1 interacts with MAPKKK18 and inhibits its activity of by promoting proteasomal degradation of MAPKKK18 (Mitula et al, 2015). These observations raise the possibility that the broad substrate specificity of PP2C (ABI1, ABI2, or HAB1) for MAPKKKs contributes to the generalized maintenance of MAPKKKs in an inactive state when lacking activation signal (e.g.…”

Convergence of Multiple MAP3Ks on MKK3 Identifies a Set of Novel Stress MAPK Modules

“…Taken together, this suggests that the pathway has two modes of functioning, which may be dependent on unidentified experimental differences between the laboratories working on this ABA-dependent MAPK pathway. Finally, a direct interaction between MAP3K18 and the PP2C phosphatase ABI1, but not with ABI2, has been described (Mitula et al, 2015). The authors hypothesized that besides being one of the main actors of the ABA core signaling module, ABI1 is also able to dephosphorylate MAP3K18 in the absence of ABA, resulting in the degradation of MAP3K18 by the proteasome.…”

“…Danquah et al (2015) characterized mutant plants in a drought and abiotic stress context and showed that plants impaired in the MKK3 module are less able to restrict water loss when submitted to a long-term mild drought experiment. More directly, the stomata of map3K18 plants are compromised to close in response to ABA (Mitula et al, 2015) whereas MAP3K18 over-expressors show accelerated leaf senescence (Matsuoka et al, 2015), a process known to be under control of ABA (Song et al, 2016). A very recent study similarly reported that Nicotiana benthamiana plants over-expressing the cotton ( Gossypium hirsutum ) GhMKK3 were more resistant to drought and closed more efficiently their stomata in response to ABA (Wang et al, 2016).…”

A Role for Barley Calcium-Dependent Protein Kinase CPK2a in the Response to Drought

“…To generate an N-terminal His-HvCPK2a fusion protein, the HvCPK2a clone was recombined with the Gateway® pDEST17™ vector (Invitrogen) using Gateway® LR Clonase® II Enzyme Mix (Invitrogen). To generate variants of HvCPK2a , a mutagenesis reaction was performed as described in Mitula et al (2015) using the following primer pairs: K94M (For 5′-GCCTGCA T GACCATCGCCAAG-3′, Rev 5′-CTTGGCGATGGTC A TGCAGGC) and D189A (For 5′-CATCCACCGCG C CCTCAAGCC-3′, Rev 5′-GGCTTGAGG G CGCGGTGGATG-3′). The vector construct 3 5S:HvCPK2a-His-GFP (Earley et al, 2006) was used for Arabidopsis protoplast transformations and transient expression assays in Nicotiana benthamiana .…”

“…Transient expression assays in Arabidopsis protoplasts or in tobacco leaves were performed as described in Ludwików et al (2014) and Mitula et al (2015). For the preparation of barley protoplasts, the central parts of 20 leaves from 6- to 10-day-old spring barley seedlings were dissected and cut into 0.5 mm strips.…”