ABA-regulated SNRK2 Protein Kinase

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Kinase names and family members

Three members of the SnRK2 family of protein kinases - SnRK2.6/OST1/SRK2E, SNRK2.2/SRK2D and SnRK2.3/SRK2I - are activated by the ABA Signaling Pathway[1][2][3][4][5][6] . SnRK2 stands for Snf1-related protein kinase family, group 2. These protein kinases have Eukaryotic Protein Kinase Catalytic Domains that are related to yeast Snf1, and they belong to the calmodulin-dependent protein kinase family of the kinome.

The best studied ABA-regulated protein kinase is SnRK2.6/OST1/SRK2E. Two of its three names originated from its membership in subclass III of the SnRK2 family of protein kinases. It was named SnRK2.6 by Hrabak et al.[7] and SRK2E by Umezawa et al.[1]. The third name OST1 (open stomata 1)[3] is descriptive of the phenotype of plants bearing a gene mutation that produces an inactive protein kinase.

In rice homologs of these protein kinases are named SAPK8, SAPK9 and SAPK10.

Kinase regulation and structure

As shown in ABA Signaling Pathway SnRK2.6/OST1/SRK2E and its homologs are regulated indirectly by ABA. Kinase activity is doubly inhibited in the absence of ABA by its binding to an ABA-regulated Protein Phosphatase 2C. This interaction results in dephosphorylation of the protein kinase's activation loop and blocking of its active site by phosphatase. When ABA binds to its receptor, the receptor binds to the protein phosphatase, freeing the protein kinase. The kinase is now free to be activated by phosphorylation of its activation loop by either itself or another protein kinase.

SnRK2.6/OST1/SRK2E has a primary structure comprising an amino terminal Eukaryotic Protein Kinase Catalytic Domain and a C-terminal sequence that contains the SNRK2 box, which is unique to the SNRK2 family and required for activity[8][9]. Its C-terminus also contains a sequence called the ABA box, which is found only in the family members that are responsive to ABA[9]

Left scene - unphosphorylated SnRK2.6 without any ligands 3uc4[8] Right scene - SnRK2.6 (blue) in complex with the protein phosphatase 2C, HAB1 (gold), with Mg2+ and SO42- 3ujg[2]

3uc4 - Apo SnRK2.6

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3uc4 scenes
The catalytic domain of SnRK2.6 is typical of Eukaryotic Protein Kinase Catalytic Domain) except for an additional α-helix (shown as strands) in the small lobe, which is formed by SNRK2 box sequence.
The activation segment (with unresolved gap), including the D of the DFG motif in ball and stick, is blue. The catalytic loop, including the D of the DLKLEN motif in ball and stick, is orchid. Subdomain III, including its invariant E in ball and stick, is gold. The invariant K of subdomain II is in chartreuse. The SnRK2 box is turquoise. The C-terminal domain, that includes the ABA box is unresolved. The arrangement of the residues in ball and stick around the active site, indicate that this structure is in a partially active state in spite of its unphosphorylated activation loop. This is possibly due to the interaction of the SNRK2 box helix with subdomain III.[8]. The interaction between these helices is similar to the interaction of helices in the complex between 1w98. Here we see that subdomain III of the protein kinase (opaque blue) is stablized by interaction with a helix from cyclin (opaque gold). The positioning of subdomain III by this interaction is critical for for formation of the active site.[10].

3ujg - SnRK2.6-HAB1

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3ujg scenes
The two enzymes are bound via interface their active sites. The phosphatase inactivates the kinase by dephosphorylating the kinase activation loop and by sterically blocking the kinase active site. The complex was constructed as a fusion protein with a 6His-tag at the N-terminus of SnRK2.6 (residues 11–362) fused to HAB1(172–511) via a GSGSAGSAAGS linker. Mutations of D296A and E297A in SnRK2.6 were introduced at the crystal packing interface to reduce surface entropy.
The same structures as in the left scene are shown. The fully resolved activation segment extends into the phosphatase's active site and is unphosphorylated. Residues 319-362 of SnRK2.6, which includes the ABA box, and the GSGSAGSAAGS linker are not resolved. The disorganization of the residues shown in ball and stick, with most pointing away from the active site, indicates that the catalytic domain is in the inactive state.
The activation loop (blue trace) of SnRK2.6 is inserted into the catalytic site (marked by the magnesium ions) of the phosphatase. The phosphorylatable residue of the activation loop S175 (CPK ball and stick) is positioned near the magnesium ions. W385 of the phosphatase (brown ball and stick) in turn protrudes into the kinase's active site, where it interacts with residues R139 and Glu144 (CPK ball and stick) of the catalytic loop (orchid trace) and I183 of the activation loop.
SnRK2.6 is shown in blue cartoon, and HAB1 in gold spacefill. The ABA box sequence is not resolved, but it would extend from the C-terminal end of the SNRK2 box helix (cyan helix). It is proposed that the ABA box sequence, which is highly acidic, binds to a patch of basic residues (blue) on the surface of the phosphatase.[2]

SNRK2 structures

3uc3 Arabidopsis thaliana SNRK2.3 + Co2+
3zut AtSNRK2.6 (D160A mutant)+ ANP
3zuu AtSNRK2.6 (D160A, S175D mutant) + gold
3uc4 apoAtSNRK2.6 (D59A, E60A mutant)
3udb apoAtSNRK2.6 (C131A, C157A, C159A, S7A, s29A, s43A, S166A, T175A)

complex with a protein phosphatase 2C
3ujg AtSNRK2.6 (D296A) + HAB1 + Mg2+


  1. 1.0 1.1 Umezawa T, Sugiyama N, Mizoguchi M, Hayashi S, Myouga F, Yamaguchi-Shinozaki K, Ishihama Y, Hirayama T, Shinozaki K. Type 2C protein phosphatases directly regulate abscisic acid-activated protein kinases in Arabidopsis. Proc Natl Acad Sci U S A. 2009 Oct 13;106(41):17588-93. doi:, 10.1073/pnas.0907095106. Epub 2009 Sep 29. PMID:19805022 doi:10.1073/pnas.0907095106
  2. 2.0 2.1 2.2 Soon FF, Ng LM, Zhou XE, West GM, Kovach A, Tan MH, Suino-Powell KM, He Y, Xu Y, Chalmers MJ, Brunzelle JS, Zhang H, Yang H, Jiang H, Li J, Yong EL, Cutler S, Zhu JK, Griffin PR, Melcher K, Xu HE. Molecular mimicry regulates ABA signaling by SnRK2 kinases and PP2C phosphatases. Science. 2012 Jan 6;335(6064):85-8. Epub 2011 Nov 24. PMID:22116026 doi:10.1126/science.1215106
  3. 3.0 3.1 Mustilli AC, Merlot S, Vavasseur A, Fenzi F, Giraudat J. Arabidopsis OST1 protein kinase mediates the regulation of stomatal aperture by abscisic acid and acts upstream of reactive oxygen species production. Plant Cell. 2002 Dec;14(12):3089-99. PMID:12468729
  4. Yoshida R, Hobo T, Ichimura K, Mizoguchi T, Takahashi F, Aronso J, Ecker JR, Shinozaki K. ABA-activated SnRK2 protein kinase is required for dehydration stress signaling in Arabidopsis. Plant Cell Physiol. 2002 Dec;43(12):1473-83. PMID:12514244
  5. Nakashima K, Fujita Y, Kanamori N, Katagiri T, Umezawa T, Kidokoro S, Maruyama K, Yoshida T, Ishiyama K, Kobayashi M, Shinozaki K, Yamaguchi-Shinozaki K. Three Arabidopsis SnRK2 protein kinases, SRK2D/SnRK2.2, SRK2E/SnRK2.6/OST1 and SRK2I/SnRK2.3, involved in ABA signaling are essential for the control of seed development and dormancy. Plant Cell Physiol. 2009 Jul;50(7):1345-63. doi: 10.1093/pcp/pcp083. Epub 2009, Jun 18. PMID:19541597 doi:10.1093/pcp/pcp083
  6. Fujii H, Verslues PE, Zhu JK. Identification of two protein kinases required for abscisic acid regulation of seed germination, root growth, and gene expression in Arabidopsis. Plant Cell. 2007 Feb;19(2):485-94. Epub 2007 Feb 16. PMID:17307925 doi:tpc.106.048538
  7. Hrabak EM, Chan CW, Gribskov M, Harper JF, Choi JH, Halford N, Kudla J, Luan S, Nimmo HG, Sussman MR, Thomas M, Walker-Simmons K, Zhu JK, Harmon AC. The Arabidopsis CDPK-SnRK superfamily of protein kinases. Plant Physiol. 2003 Jun;132(2):666-80. PMID:12805596 doi:10.1104/pp.102.011999
  8. 8.0 8.1 8.2 Ng LM, Soon FF, Zhou XE, West GM, Kovach A, Suino-Powell KM, Chalmers MJ, Li J, Yong EL, Zhu JK, Griffin PR, Melcher K, Xu HE. Structural basis for basal activity and autoactivation of abscisic acid (ABA) signaling SnRK2 kinases. Proc Natl Acad Sci U S A. 2011 Dec 27;108(52):21259-64. Epub 2011 Dec 12. PMID:22160701 doi:10.1073/pnas.1118651109
  9. 9.0 9.1 Belin C, de Franco PO, Bourbousse C, Chaignepain S, Schmitter JM, Vavasseur A, Giraudat J, Barbier-Brygoo H, Thomine S. Identification of features regulating OST1 kinase activity and OST1 function in guard cells. Plant Physiol. 2006 Aug;141(4):1316-27. Epub 2006 Jun 9. PMID:16766677 doi:pp.106.079327
  10. Honda R, Lowe ED, Dubinina E, Skamnaki V, Cook A, Brown NR, Johnson LN. The structure of cyclin E1/CDK2: implications for CDK2 activation and CDK2-independent roles. EMBO J. 2005 Feb 9;24(3):452-63. Epub 2005 Jan 20. PMID:15660127

See Also

[1] Abscisic Acid in Wikipedia

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Alice Harmon, Michal Harel

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