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SnRK2.6/OST1/SRK2E

Role in Abscisic Acid signaling

SnRK2.6/OST1/SRK2E is a triply-named protein kinase from Arabidopsis, which is activated by the abscisic acid (ABA) response pathway[1][2][3][4]. As shown in the figure, in unstimulated cells SnRK2.6/OST1/SRK2E (K) and a protein phosphatase 2C (P) are in a complex (K-P) in which the kinase is dephosphorylated and inactive[5][6]. The kinase and phosphatase are proposed to be tethered via the C-terminal sequence (dashed line) of the kinase[5][6][7]. When ABA levels in the cytosol rise, ABA binds to an ABA receptor (R)[8][9][10]. The activated receptor (R.ABA) binds to the protein phosphatase (R.ABA-P) and inactivates it. SnRK2.6/OST1/SRK2E, although still tethered to the phosphatase, is now free to be activated by autophosphorylation or phoshorylation by another protein kinase[11][12]. Activation of SnRK2.6/OST1/SRK2E leads to phosphorylation of: 1) ion channels SLAC1[13] and KAT1[14] in guard cells and stomatal closure; 2) transcription factor ABI5[3] in seeds/seedlings and dormancy/growth arrest; or 3) phosphorylation of transcription factor AREB/ABF [15][4] in vegetative tissue and stress tolerance and growth regulation.

Structures in the figure are: R, apo pYR2, 3kdh; R.ABA, PYR2.ABA, 3kdi; K-P; SnRK2.6-HAB1, 3ujg; R.ABA-P, PYR2.ABA-ABI2, 3ujl; K, SnRK2.6,3uc4.

Kinase names and family members

Two of SnRK2.6/OST1/SRK2E's 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.[16] and SRK2E by Umezawa et al.[5]. SnRK2 stands for SNF1-related kinase group 2, which in Arabidopsis has 10 members. SNRK2s are members of the calmodulin-dependent protein kinase clade of protein kinases. The third name OST1 (open stomata 1)[1] is descriptive of the phenotype of plants bearing a gene mutation that produces an inactive protein kinase.

Two other family members in Arabidopsis, SNRK2.2/SRK2D and SnRK2.3/SRK2I, are activated by the ABA pathway in the same manner as SnRK2.6. Each of these kinases interacts with a member of clade A of the protein phosphatase 2C family - ABI1, HAB1 or HAB2. In rice homologs of these protein kinases are named SAPK8, SAPK9 and SAPK10.

Kinase structure and regulation

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[17][18]. Its C-terminus also contains a sequence called the ABA box, which is found only in the family members that are responsive to ABA[18]

Left scene - unphosphorylated SnRK2.6 without any ligands 3uc4[17] Right scene - SnRK2.6 (blue) in complex with the protein phosphatase 2C, HAB1 (gold), with Mg2+ and SO42- 3ujg[6]

3uc4 - Apo SnRK2.6


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.[17]. 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.[19].









3ujg - SnRK2.6-HAB1


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.[6]


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+


References

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See Also

[1] Abscisic Acid in Wikipedia

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Alice Harmon

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