User:Alice Harmon/Sandbox 4: Difference between revisions

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|'''Left scene''' - The crystal structure [[3uc4]] shows that 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. This helix parallels subdomain III and resembles **blah blah** kinase structures.  
|'''Left scene''' - The crystal structure [[3uc4]] shows that 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. This helix parallels subdomain III and resembles **blah blah** kinase structures.  
<br>
<br>
|'''Right scene''' - The crystal structure [[3ujg]] shows the SnRK2.6–HAB1 complex, which was constructed as a fusion protein with a H6-tag (MAHHHHHHA) at the N-terminus of SnRK2.6 (residues 11–362) fused to HAB1(172–511) with a GSGSAGSAAGS linker. Residues 319-362 of SnRK2.6, which includes the ABA box, and the linker sequence are not resolved in the 3D structure.  D296A and E297A are mutations introduced at the crystal packing interface to reduce surface entropy.  
|'''Right scene''' - The crystal structure [[3ujg]] shows the SnRK2.6–HAB1 complex, which was constructed as a fusion protein with a H6-tag (MAHHHHHHA) at the N-terminus of SnRK2.6 (residues 11–362) fused to HAB1(172–511) with a GSGSAGSAAGS linker.  D296A and E297A are mutations introduced at the crystal packing interface to reduce surface entropy.  
|-
|-
| <applet load='3ujg' size='400' frame='true' align='left' caption='3uc4 - Apo SnRK2.6' scene = '55/559985/Aposnrk2_6/6' /><br  clear='both'>'''3uc4 scenes''' <Br><scene name='55/559985/Aposnrk2_6/6'>1. Default scene</scene><Br><scene name='55/559985/Aposnrk2_6critical/1'>2. Important structures</scene>Activation loop (with gap) in blue<br>
| <applet load='3ujg' size='400' frame='true' align='left' caption='3uc4 - Apo SnRK2.6' scene = '55/559985/Aposnrk2_6/6' /><br  clear='both'>'''3uc4 scenes''' <Br><scene name='55/559985/Aposnrk2_6/6'>1. Default scene</scene><Br><scene name='55/559985/Aposnrk2_6critical/1'>2. Important structures:</scene> 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 pointing toward the active site, indicate that this structure is in the active state.  
Catalytic loop in orchid<br>
| <applet load='3ujg' size='400' frame='true' align='left' caption='3hx4 - active TgCDPK1' scene = '55/559985/Aposnrk2_6/2' /><Br clear='both'>'''3ujg scenes'''<Br><scene name='55/559985/Aposnrk2_6/2'>1. Default Scene</scene><br><scene name='55/559985/Ost1hab1_critical/3'>2. Important structures</scene>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 in ball and stick, with most pointing away from the active site, indicates that the catalytic domain is in the inactive state. 
C-helix in yellow<br>
<br><Br><scene name='55/559985/Ost1hab1_interaction/1'>3. Zone of interaction </scene><br>
K in chartreuse<br>
phosphatase W385 in brown. The phosphatase is sterically blocking the kinase active site, in addition to in activating the kinase by dephosphorylating the kinase activation loop.
Snrk2 box (unique to Snrk's) in turquoise.<br>
C-terminal acidic domain missing.<br><br><br>
| <applet load='3ujg' size='400' frame='true' align='left' caption='3hx4 - active TgCDPK1' scene = '55/559985/Aposnrk2_6/2' /><Br clear='both'>'''3ujg scenes'''<Br><scene name='55/559985/Aposnrk2_6/2'>1. Default Scene</scene> <Br><scene name='55/559985/Ost1hab1_interaction/1'>2. Zone of interaction </scene><br><scene name='55/559985/Ost1hab1_critical/3'>2. Important structures</scene>
kinase activation loop in blue<br>
phosphatase W385 in brown
|}
|}


Revision as of 16:58, 1 October 2013

SnRK2.6/OST1/SRK2ESnRK2.6/OST1/SRK2E

Role in Abscisic Acid signalingRole 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 in plants[1][2][3][4]. As shown in the figure, in unstimulated cells SnRK2.6/OST1/SRK2E (K in the figure) and a protein phosphatase 2C (ABI1 or its homologs; P in the figure) 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 (PYR1/PYL/RCAR; R in the figure)[8][9][10]. The activated receptor (R.ABA) binds to the protein phosphatase (R.ABA-P) and inactivates it. SnRK2.6/OST1/SRK2E is still tethered to the phosphatase, but its active site 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 membersKinase 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 regulationKinase 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]. Its C-terminus also contains a sequence called the ABA box, which is found only in the family members that are responsive to ABACite error: Closing </ref> missing for <ref> tag

Left scene - The crystal structure 3uc4 shows that 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. This helix parallels subdomain III and resembles **blah blah** kinase structures.


Right scene - The crystal structure 3ujg shows the SnRK2.6–HAB1 complex, which was constructed as a fusion protein with a H6-tag (MAHHHHHHA) at the N-terminus of SnRK2.6 (residues 11–362) fused to HAB1(172–511) with a GSGSAGSAAGS linker. D296A and E297A are mutations introduced at the crystal packing interface to reduce surface entropy.

3uc4 - Apo SnRK2.6

Drag the structure with the mouse to rotate

3uc4 scenes

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 pointing toward the active site, indicate that this structure is in the active state.

3hx4 - active TgCDPK1

Drag the structure with the mouse to rotate

3ujg scenes

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 in ball and stick, with most pointing away from the active site, indicates that the catalytic domain is in the inactive state.




phosphatase W385 in brown. The phosphatase is sterically blocking the kinase active site, in addition to in activating the kinase by dephosphorylating the kinase activation loop.




ABA box is not seen in the crystal structure.

Clearest picture of Pyr1 gate - latch is in Annu Reviews article


unused:


SNRK2 structuresSNRK2 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+



PP2C structuresPP2C structures

1a6q – hPP2C
2iq1 – hPP2C κ
2p8e – hPP2C β
2cm1 – PP2C + Mn – Micobacterium tuberculosis
3d8k – PP2C – Toxoplasma gondii
3jrq, 3kdj, 3nmn – AtPP2C + Pyl1 – Arabidopsis thaliana
3nmt, 3kb3, 3nmv, 3ujl – AtPP2C + Pyl2
4ds8 – AtPP2C + Pyl3 + Mn
3rt0 – AtPP2C (mutant) + Pyl10
3qn1, 3zvu – AtPP2C + Pyr1
3ujg – AtPP2C + SNRK2
3ujk – AtPP2C


ReferencesReferences

  1. ↑ 1.0 1.1 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
  2. ↑ 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
  3. ↑ 3.0 3.1 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
  4. ↑ 4.0 4.1 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
  5. ↑ 5.0 5.1 5.2 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
  6. ↑ 6.0 6.1 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
  7. ↑ Yoshida R, Umezawa T, Mizoguchi T, Takahashi S, Takahashi F, Shinozaki K. The regulatory domain of SRK2E/OST1/SnRK2.6 interacts with ABI1 and integrates abscisic acid (ABA) and osmotic stress signals controlling stomatal closure in Arabidopsis. J Biol Chem. 2006 Feb 24;281(8):5310-8. Epub 2005 Dec 19. PMID:16365038 doi:M509820200
  8. ↑ Yin P, Fan H, Hao Q, Yuan X, Wu D, Pang Y, Yan C, Li W, Wang J, Yan N. Structural insights into the mechanism of abscisic acid signaling by PYL proteins. Nat Struct Mol Biol. 2009 Dec;16(12):1230-6. Epub 2009 Nov 5. PMID:19893533 doi:10.1038/nsmb.1730
  9. ↑ Ma Y, Szostkiewicz I, Korte A, Moes D, Yang Y, Christmann A, Grill E. Regulators of PP2C phosphatase activity function as abscisic acid sensors. Science. 2009 May 22;324(5930):1064-8. doi: 10.1126/science.1172408. Epub 2009, Apr 30. PMID:19407143 doi:10.1126/science.1172408
  10. ↑ Park SY, Fung P, Nishimura N, Jensen DR, Fujii H, Zhao Y, Lumba S, Santiago J, Rodrigues A, Chow TF, Alfred SE, Bonetta D, Finkelstein R, Provart NJ, Desveaux D, Rodriguez PL, McCourt P, Zhu JK, Schroeder JI, Volkman BF, Cutler SR. Abscisic acid inhibits type 2C protein phosphatases via the PYR/PYL family of START proteins. Science. 2009 May 22;324(5930):1068-71. doi: 10.1126/science.1173041. Epub 2009, Apr 30. PMID:19407142 doi:10.1126/science.1173041
  11. ↑ Boudsocq M, Droillard MJ, Barbier-Brygoo H, Lauriere C. Different phosphorylation mechanisms are involved in the activation of sucrose non-fermenting 1 related protein kinases 2 by osmotic stresses and abscisic acid. Plant Mol Biol. 2007 Mar;63(4):491-503. PMID:17103012 doi:10.1007/s11103-006-9103-1
  12. ↑ Burza AM, Pekala I, Sikora J, Siedlecki P, Malagocki P, Bucholc M, Koper L, Zielenkiewicz P, Dadlez M, Dobrowolska G. Nicotiana tabacum osmotic stress-activated kinase is regulated by phosphorylation on Ser-154 and Ser-158 in the kinase activation loop. J Biol Chem. 2006 Nov 10;281(45):34299-311. Epub 2006 Sep 15. PMID:16980311 doi:10.1074/jbc.M601977200
  13. ↑ Geiger D, Scherzer S, Mumm P, Stange A, Marten I, Bauer H, Ache P, Matschi S, Liese A, Al-Rasheid KA, Romeis T, Hedrich R. Activity of guard cell anion channel SLAC1 is controlled by drought-stress signaling kinase-phosphatase pair. Proc Natl Acad Sci U S A. 2009 Dec 15;106(50):21425-30. doi:, 10.1073/pnas.0912021106. Epub 2009 Dec 2. PMID:19955405 doi:10.1073/pnas.0912021106
  14. ↑ Sato A, Sato Y, Fukao Y, Fujiwara M, Umezawa T, Shinozaki K, Hibi T, Taniguchi M, Miyake H, Goto DB, Uozumi N. Threonine at position 306 of the KAT1 potassium channel is essential for channel activity and is a target site for ABA-activated SnRK2/OST1/SnRK2.6 protein kinase. Biochem J. 2009 Dec 10;424(3):439-48. doi: 10.1042/BJ20091221. PMID:19785574 doi:10.1042/BJ20091221
  15. ↑ Furihata T, Maruyama K, Fujita Y, Umezawa T, Yoshida R, Shinozaki K, Yamaguchi-Shinozaki K. Abscisic acid-dependent multisite phosphorylation regulates the activity of a transcription activator AREB1. Proc Natl Acad Sci U S A. 2006 Feb 7;103(6):1988-93. Epub 2006 Jan 30. PMID:16446457 doi:10.1073/pnas.0505667103
  16. ↑ 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
  17. ↑ 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
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