Structural and functional studies of Arabidopsis SIZ1(Cheong et al., 2009)

Specific domain structures control ABA, SA, and stress mediated SIZ1 phenotypes

Background

PART 2

Structural Domain of SIZ1 and its point mutations

SAPLxxVL

PHDC3CHC3

PINITPIIT

SP-RINGCCHC3

SXShhxSxSaaaSIZ1WT

SAPLxxAA

SIZ1sap

PHDC3YHC3

SIZ1phd

SP-RINGCAHC3SIZ1sp-ring

PINITPAATSIZ1pinit

SXShhxAxAaaaSIZ1sxs

The PHD domain of SIZ1 may have a plant specific role involving light and sugar signaling

The PHD mutation(C134Y) is shown long hypocotyl elongation

SIZ1

phd

SIZ1

WT

Col

-0siz1-2

3% D-GlucoseCol

-0SI

Z1/ S

IZ1

phd

(ph

d C

ol-o )

2% Sucrose

The mutation of SIZ1 PHD domain dominantly and negatively works for hypocotyls elongation. When carbohydrate was sup-plied to seedling early growth, the hypocotyls from mutant (SIZ1phd) are longer than wild type. This phenotype was represen-tative even Col-0 wild type background (phdcol-0) which has SIZ1 protein (wild type) is normally expressed. The seedlings are 7 days old and grown under 16 hr light/8 hr dark at 22 using indicated carbohydrate. One another thing, this phenotype is ℃not shown under constitutive dark (next slide)

0% Sugar

2% Sucrose

Light Dark

The hypocotyl phenotype is dependent on light and sugar

Catala et al., 2007

SIZ1 is expressed in all tissues and all developmental stage but not in hypocotyl.

At5g60410 (SIZ1)At5g60400

β-Glucoronidase (GUS)

SIZ1 promoter (3650bp)

Dominant and Negative effect of C134Y(phd mutation)

All seeds from phdCol-0 plants exhibit bright color both heterozygote (T1 generation) and homozygote (since T2 generation).All plants are grown under the same condition at the same time. Seeds were harvested from the matured plants. Photo was taken T3 seeds which harvested a pool from T2 plants. The number from 1 to 4 are indicated individual T1 line.

PHY A (or PHY B) may be regulated by PHD region of SIZ1

Background :: 1. the KO mutant of these photo-receptor are exhibit a long hypocotyl and bright seed color .2. These proteins are formed the same structural feature, nuclear body.3. These proteins are regulated by post-translational modification such as ubiqutination, phosphorylation.

The Scenario>>>

SIZ1(PHD) PHYA/PHYB activity

Subcellular localizationProtein stabilityAccelerating other modification (such as conformational change or dimerization)

If the PHD region of SIZ1 works as a scaffold protein of PHYA, PHYA’s activity may be regulated by SIZ1 due to either SUMOylation or not.

Alternatively, the PHYA(PHYB) target proteins such as PKSs can be also regulated by SIZ1

3. Determine sub-cellular localization and interaction between SIZ1 and PHY A.

; BiFC (or Y2H) between SIZ1 and PhyA(wild type and sumoylation site mutants) . ; identify the interaction domain with SIZ1 or SUMOylation site of PhyA.

1.Determine the phenotype of double mutation at K100/K488

; To observe the morphological phenotypes of transgenic plants including the possibility of epigenetic trait; To test SUMO binding and auto-sumoylation. ; To test SUMO E3 ligase activity

Study plan and Ongoing works

4. Identify the relationship between SIZ1 regulation modules(activity, stability) and sumoylation of target protein such as PhyA. ; hypocotyl phenotype investigation using double(or triple) mutants such as phyA X siz1-2 (X SIZ1phd)

2. Determine the hypocotyl phenotype of transgenic plants, SIZ1dphd which has no PHD domain like mammalian PIAS s or yeast SIZ1. ;SIZ1dphd (T2 plants and waiting for T3 seeds for homozygote pool.) >> T1 and T2(homozygote) plants are the same morphological phenotype to siz1-2 mutant. >> Determine SIZ1phd (C134Y) is either a gain of function mutant (neotropism) or a regulator

AtSOS1 C-term can be phosphorlation by MPK3

PART 3

AtSOS1 C-term protein can be phosphorlation by MPK3

MPK3

SOS1 C-term

Ca2+

+

++

++-

+-

-+-

+

+

--

(a positive control for MPK sub-

strate) MBP

1. Putative MPK phosphorlyation sites in SOS1 C-terminus part are S925, S930, and S1136 . In addition, S1138 could be MPKs phosphorylation site as well as SOS2 phosphorylation site.

Identify the phosphorylation site by kinase assay using mutant proteins.

3. Interaction test with MPKs and SOS1

2. Biological function investigation of SOS1 phosphorylation by MPK3 using null mutant both mpk3 and sos2 after designing the constitutively phosphorylated and (or) unphosphorylated form from #1 result.

such as responsiveness under the salt stress.

By Prof. Chung lab.

MPK3

SOS1-C term

SOS1 several mutants are still phosphoylated by MPK3

MP

K6

MP

K4

WT

SO

S1-

CD

2

GS

T

MPK3

SOS1-C

S98

6A

WT

S10

54A

S11

36A

S11

38A

S11

36/

1138

A

WT

GST

SOS1-CD2

SOS1-C

*

In vitro kinase assay Recombinant proteins, wild type (WT) and mutant (S986A, S1054A, S1136A, S1138A, S1136/1138A) are purified from E.coli. GST was used as a negarive control. Wild type and mutants are still phosphorylated by MPK3 but MPK4 and MPK 6 are not phosphorylated even WT. Upper panel is the scheme of the recombinant protein of SOS1. Only cloned SOS1-CD2 and tested phosphorylation. The result indicated that there was no phosphorylation in the CD2 fragment. (* is un-washed E-Coli protein during purification and is phosphorylated ). In case of S1054A, the protein used smaller than other and showed lower intensity . In case of S1136 A and S1138A are used the same amount to WT and S986A and showed lower intensity. This is speculated that one of these should be a phosphorylation site. In addition, another phosphorylation site should be predicted . To consider these points, other possible sites are doing site directed mutageneses and making double or triple mutants.

GSTGSTGSTGST SOS1-C

SOS1-CD1 SOS1-CD2SOS1-CD3

454 611454 1146*

1146*610 857

855

AtSOS1 protein can interact with MPK3(?)

SOS1 MPKs

If interaction,

MPKs>> MPK1, MPK2, MPK3, MPK4, MPK5, MPK6, MPK9

To show that

Still now, Nub-SOS1 C-term was cloned and is doing a transformation using yeast cell. After selecting the trans-formed cells, the growth assay will be done. Like this, the growth assay will be confirmed using SOS1 full form clone..

Thank You