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TitleRhoファミリー低分子量G蛋白質特異的グアニンヌクレオチド交換因子PLEKHG2/FLJ00018の新規活性制御機構とその細胞機能( 本文(Fulltext) )

Author(s) 佐藤, 克哉

Report No.(DoctoralDegree) 博士(薬科学) 連創博甲第24号

Issue Date 2014-03-25

Type 博士論文

Version ETD

URL http://hdl.handle.net/20.500.12099/49052

※この資料の著作権は、各資料の著者・学協会・出版社等に帰属します。

RRho G

PLEKHG2/FLJ00018

Cellular functions of novel regulation mechanisms of FLJ00018, a Rho familly small G-protein-specific guanine nucleotide exchange factor

2014

1

1 FLJ00018 EGF

1 7

2

1. 9

2. 10

3. Serum response element (SRE ) 10

4. FLJ00018 10

5. 11

6. Mn2+ Phos-tag SDS-PAGE 11

7. 12

8. 12

3

1. FLJ00018 1 13

2. FLJ00018 1AR EGF transactivation 14

3. FLJ00018 EGF 17

4. FLJ00018 H-Ras 20

5. FLJ00018 H-Ras/MAPK 21

6. EGF FLJ00018 23

7. EGF FLJ00018 25

8. EGF FLJ00018 27

4 30

2 Src

1 34

2

1. 36

2. 36

3. SH2 36

4. 37

5. 37

3

1. FLJ00018 cSrcCA 39

2. cSrcCA FLJ00018 39

3. FLJ00018 489

41

4. FLJ00018 ABL1 PIK3R3 42

5. Abl1 SrcCA FLJ00018 42

6. EphB2/Src FLJ00018 43

4 46

3 FLJ00018

1 48

2

1. 50

2. two-hybrid 50

3. 51


5. 51

6. Pull-down 52

7. 52

3

1. FLJ00018 53

2. FLJ00018 - - 54

3. - FLJ00018 55

4. - - FLJ00018

SRE 57

5. - FLJ00018 G 60

6. FLJ00018 60

4 63

4 FLJ00018 Four and a half LIM 1 (FHL1)

1 65

2

1. 67

2. 67


4. 67

5. 68

3

1. FLJ00018 Four and a half LIM domain1 (FHL1)

69

2. FLJ00018 FHL1 69

3. FHL1 FLJ00018 71

4. FHL1 FLJ00018 P2 71

5. FHL1 FLJ00018 SRE 73

6. FHL1 FLJ00018 G 75

7. FHL1 FLJ00018 SRE 76

8. FLJ00018 FHL1 77

4 82

85

89

89

98

101

1

60

2 1

gain of function

loss of function

(RTK)

(EGFR) RTK

EGFR

gain of function (1-3)

2

(GPCR) 2% 800

GPCR (4) GPCR

(G ) G

G G

(5) G Gs, Gi,

Gq/11, G12/13 4 20 G ,

G 5 14

(Fig. 1) G GDP GTP

GPCR GPCR GDP GTP

Fig. 1 G

3

APC

K-Ras

APC K-Ras

p53

(Fig. 2) (6)

K-Ras Ras GTP (small GTPase)

small GTPase GTP GDP

GDP GTP GDP/GTP

(GEF) GTPase (GAP)

(GDI) GEF GDP GTP

GAP Small GTPase GTP GDP GTPase GDI GDP

GTPase (Fig. 3) Ras

small GTPase Ras, Rho, Rab, Ran, Arf 5

Ras

Rho Rab

Ran Arf (Fig. 4)

Fig. 22

4

GPCR Small GTPase

(Fig. 5) Rho

Small GTPase (RhoGTPase)

GPCR RhoGTPase

Rac (7) RhoGTPase

23 RhoA, Rac1, Cdc42 (8)

RhoA

Fig. 33 SSmall GTPase

Fig. 44 SSmall GTPase

5

Rac1 (lamellipodia) Cdc42

(filopodia)

(9) (Fig. 6) RhoGTPase GDP GTP

RhoGTPase GEF (RhoGEF) (10) RhoGEF 2

1 GEF Dbl homology domain (DH

) Pleckstrin homology domain (PH ) Dbl

Dbl RhoGEF 70

(11,12) RhoGEF 1 DH PH

Dock homology region (DHR) 2 CDM-zizimin homology (CZH)

11 (13) Dbl RhoGEF

leukemia-associated RhoGEF (LARG), P115-RhoGEF, PDZ-RhoGEF DH

PH Regulator of G-protein signaling domain (RGS )

RGS G G 12/13

RhoA GDP/GTP (14-16) p63-RhoGEF Trio, Karilin

RhoGEF G Gq

(17-19) G G RhoGEF P-Rex1

(20) P-Rex1 Rac

P-Rex1

(21)

Fig. 55 G RhoGEF Rho

6

PLEKHG2/FLJ00018

Evi24 Dbl

RhoGEF (22) Dbl RhoGEF

FLJ00018 N DH PH Ueda

FLJ00018 DH N GPCR G

G

(23) FLJ00018 RhoGTPase Rac1, Cdc42

jurkat T FLJ00018 G

(24) RhoGEF

FLJ00018

1386

FLJ00018 DH PH

DH, PH C

FLJ00018

Fig. 66 RRhoGTPase

7

1 FLJ00018 EGF

1

RhoGEF GPCR

RGS RhoGEF p115-RhoGEF LARG, PDZ-RhoGEF

G 13 RGS RhoA GEF (14)

PDZ-RhoGEF N PSD-95/Dlg/Zo-1 domain (PDZ )

LPA RhoA (25) PDZ-RhoGEF

RhoGEF G G q PDZ

RhoA (26)

RhoGEF

70 RhoGEF 19

(27) PIX Vav3, Asef RhoGEF

Asef EGFR Rac1,

Cdc42 (28) PIX (FGFR)

/ (29)

p63-RhoGEF 23, 25 26

RhoA

(30)

RhoGEF GPCR RhoGTPase

RhoGEF GPCR GPCR

RhoGEF

FLJ00018 G

8

FLJ00018 FLJ00018

9

2

1.

pcDNA3.1- 1- ( 1AR), pcDNA3.1-G 1, pcDNA3.1-G 2,

pcDNA3.1-H-Ras AC (H-Ras ), pcDNA3.1-H-Ras T17N (H-Ras

), pcDNA3.1-RhoA T19N, pcDNA3.1-Rac1 T17N, pcDNA3.1-Cdc42

T17N, pCMV5 Missouri S&T cDNA Resource Center

PCR pF4A-CMV pF5A-CMV-neo pFN21A

pCMV5- ARKct

pFC21A-Halo-EGFR, pFN21K-Halo-Vav3 DNA FLJ00018

cDNA DNA

(31) FLJ00018 FLJ00018 ORF Gateway

(Life Technologies) pcDNA3.1-DEST47

pcDNA3.1-DEST47-FLJ00018 PCR pFN21A-Myc

pFN21K-Halo FLJ00018

pFN21A-Myc-FLJ00018 PCR FLJ00018

FLJ00018 T680A pFN21A-Myc-FLJ00018 PCR

(Stratagene) (AG ; Azami green)

(32)

phmAG1-MCLinker MBL

PCR pFN21A-mAG-FLJ00018 pSRE.L-luciferase

reporter Stratagene pRL-SV40 Nippon Gene Isoproterenol

KN-93 SB202190 Calbiochem, EGF Peprotech AG1478, U0126

Wako SP600125 Sigma phosphase inhibitor

protease inhibitor Roche Lambda phosphatase

New England Biolabs

10

22.

NIH3T3 37 , 5% CO2 10% calf serum (CS)

(DMEM) HEK293 Neuro-2a 37 , 5% CO2

10% Fetal Bovine Serum (FBS) DMEM

Lipofectamine-Plus (Life

Technologies) serum response element (SRE) 0.4 g DNA

1 g DNA 2 g DNA

3 1× insulin-transferrin-selenium-X (Invitrogen)

6-8 16-18

3. Serum response element (SRE )

24 pRL-SV40

pSRE.L-luciferase

PBS 1

13200 rpm, 10 min

dual luciferase reporter assay system (Promega)

±S.D.

4. FLJ00018

PBS (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.5% Nonidet

P-40, protease inhibitor) 13200 rpm, 10 min

10 unit 400 unit Lambda phosphatase

30 2 2× SDS sample buffer

11

55.

PBS 1% (w/v) SDS / 20 mM Tris-HCl / 1 mM EDTA

(pH 8.0) 1×SDS sample buffer

BCA protein assay kit (Thermo Scientific)

7.5% 10% SDS-

(SDS-PAGE) PVDF 3%

5% Bovine serum albumin (BSA) TBS-T (Tris buffered saline ; 20

mM Tris-HCl, pH7.5, 0.15 M NaCl, 0.01% NP-40) Myc-

FLJ00018 1:2000 Anti-Myc (Roche, 0.2 g/ml)

1:5000 horseradish peroxidase (HRP)-

IgG (MBL) Halo- FLJ00018 Vav3

1:2000 Anti-Halo (Promega, 0.2 g/ml)

1:5000 HRP- IgG (MBL)

EGFR 1:2000 Anti-EGFR (Santa Cruz, 1000

g/ml) 1:5000 HRP- IgG

1:2000 Anti-P-Tyr (PY20)

(Santa Cruz Biotechnology, stock 100 g/ml) 1:5000

HRP- IgG

enzyme-linked chemiluminescence (GE Healthcare) LAS-4000 image

analyzer (Fuji Film)

6. Mn2+ Phos-tag SDS-PAGE

FLJ00018

Mn2+ Phos-tag AAL107 SDS

12

(33) AAL107

PBS 1% (w/v) SDS H2O

1× SDS sample buffer

BCA protein assay kit (Thermo Scientific) FLJ00018

(FLJ00018 WT)

(34,35) 13 M Phos-tag AAL107 (Wako), 26 M MnCl2, 1.5% 3%

SDS-PAGE FLJ00018 15 M Phos-tag AAL107

(Wako), 25 M MnCl2, 6% SDS- 1 mM EDTA

10 Mn2+

7.

PBS 30 4%

Perma Fluor

(BZ-9000; KEYENCE) sample

10 50

Image J

8.

3 ±S.D.

student-T-test P

13

3

1. FLJ00018 1

FLJ00018 m2 G

(23) FLJ00018 GPCR

1AR FLJ00018 NIH3T3 Rho

GTPase SRE (36) 1AR G s

G s 1AR

G

FLJ00018 1AR AR Isoproterenol (20 M)

(Fig. 1-1B)

Fig. 1--1 NIH3T3 FLJ00018 SRE Issoproterenol-ARKct

A, NIH3T3 pSRE.L-luciferase, pRL-SV40 DNA, FLJ00018, -ARKct, AR

20 M, 5.5 Isoproterenol dual-luciferase reporter assay system Mock 1.0 3

± S.D. B, NIH3T3 1AR Myc FLJ00018

Isoproterenol (20 M) 157.5% SDS-PAGE Anti-Myc

A B

14

SRE (Fig. 1-1A) C-terminal of -adrenergic

receptor kinase ( -ARKct) -ARKct

G G G

-ARKct Isoproterenol SRE -ARKct

SRE G

1AR G FLJ00018

22. FLJ00018 1AR EGF transactivation

GPCR EGFR transactivation

(37-42) transactivation GPCR 1AR 2AR

GPCR EGFR EGFR

EGFR

NIH3T3 EGFR 1AR FLJ00018 Isoproterenol

(20 M)

A B

15

C D

E F

G

Fig. 11-22 FLJ00018 1AR EEGF ttransactivation

A, NIH3T3 EGFR, 1AR Myc FLJ00018Isoproterenol (20 M)

0-30 7.5% SDS-PAGEAnti-P-Tyr (PY20) Anti-EGFR B, NIH3T3 pSRE.L-luciferase, pRL-SV40 DNA, FLJ00018, AR, EGFR

EGFRAG1478 (10 M, 1 ) 20 M, 5.5 Isoproterenol

dual-luciferase reporter assay systemMock 1.0 3 ± S.D.#, p

16

Isoproterenol EGFR 15

1AR EGFR transactivation

(Fig. 1-2A) Isoproterenol FLJ00018

EGFR EGFR

AG1478 SRE FLJ00018 1AR EGFR NIH3T3

Isoproterenol (20 M) SRE

AG1478 (10 M) Isoproterenol (10 M) SRE

(Fig. 1-2B) FLJ00018

Isoproterenol FLJ00018

(Fig. 1-2C) G FLJ00018

EGFR transactivation FLJ00018

Vav Asef, Tiam-1, -Pix

RhoGEF RhoGEF

RhoGEF (27,28,43) FLJ00018 EGFR

transactivation

Lambda

Isoproterenol

Fig. 11-22 D, NIH3T3 EGFR, 1AR Myc FLJ00018

Isoproterenol (20 M) 15 (10 unit)

Lambda (400 unit) 30 2 SDS

F, HEK293 EGFR, 1AR Myc FLJ00018

Isoproterenol (20 M) 15 13 M Phos-tagAAL107, 26 M MnCl2, 1.5%

3% SDS-PAGE Myc FLJ00018Anti-Myc

17

Isoproterenol (Fig. 1-2D)

Phos-tag

Phos-tag Mn2+ Phos-tag AAL107

AAL107

(33-35)

Isoproterenol HEK293 Phos-tag


(Fig. 1-2E) FLJ00018 (Fig. 1-2F) FLJ00018

EGFR transactivation AG1478


AG1478 (Fig. 1-2G) FLJ00018 EGFR

transactivation

33. FLJ00018 EGF

Figure 1-2 FLJ00018 GPCR EGFR transactivation

EGF EGFR FLJ00018

EGFR FLJ00018 NIH3T3 SRE

6 EGF (20 ng/ml) SRE

(Fig. 1-3A) EGF FLJ00018 RhoGTPase

RhoGTPase

SRE G

RhoA EGFR FLJ00018 SRE

Rac1 Cdc42

EGFR FLJ00018 SRE (Fig. 1-3B)

18

EGF FLJ00018 G Rac1, Cdc42

EGF FLJ00018 EGF (20 ng/ml)

FLJ00018 (Fig. 1-3C)

Lambda

Lambda

EGF FLJ00018 (Fig.

1-3D) Phos-tag HEK293 EGF FLJ00018

FLJ00018 (Fig. 1-3E, F) FLJ00018

EGF

EGF FLJ00018 FLJ00018 EGFR

EGF

FLJ00018 EGF 2 15 5

(Fig. 1-3G) 1AR EGFR (Fig. 1-2E) EGF

FLJ00018

EGFR

FLJ00018 EGFR Figure 1-3H

Vav3 EGFR EGF Vav3

EGFR FLJ00018 EGF FLJ00018

EGFR FLJ00018

20

4. FLJ00018 H-Ras

EGF FLJ00018

EGFR Ras-MAPK

(38) NIH3T3 FLJ00018 H-Ras (H-Ras CA)

H

Fig. 11-33 FFLJ00018 EEGF A, B, NIH3T3 pSRE.L-luciferase, pRL-SV40 DNA, FLJ00018, EGFR, pF4A-RhoA T19N, pF4A-Rac T17N, pF4A-Cdc42 T17N

20 ng/ml, 6 EGFdual-luciferase reporter assay system Mock

1.0 3 ± S.D. C, E, G, H, NIH3T3 (C, G, H) HEK293 (E) EGFR, G 1 2 Myc

Halo FLJ00018, Halo Vav3EGF (20 ng/ml)

15 (C, E, H) (G) 7.5% SDS-PAGE Myc FLJ00018 Anti-Myc Halo

Anti-HaloAnti-P-Tyr (PY20) D, NIH3T3 EGFR Myc FLJ00018

20 ng/ml EGF (15) (10 unit) Lambda

(400 unit) 30 2 SDS

F, HEK293 EGFR Myc FLJ0001820 ng/ml EGF (15

) 13 M Phos-tag AAL107, 26 M MnCl2, 1.5% 3% SDS-PAGE Myc FLJ00018Anti-Myc

21

H-Ras (H-Ras DN)

H-Ras CA FLJ00018 (Fig. 1-4A)

H-Ras DN EGF (20 ng/ml) FLJ00018

(Fig. 1-4B) H-Ras EGF FLJ00018

5. FLJ00018 H-Ras/MAPK

EGFR-H-Ras MAPK MAPK

MEK U0126 (10 M) 30 EGF

(20 ng/ml, 15 ) FLJ00018

(CaMK ) KN-93 (10 M)

EGF FLJ00018 U0126 (10 M)

EGF (Fig. 1-5A)

FLJ00018 MEK

U0126 (10 M, 1 ) KN-93 (10 M, 1 )

SRE EGF SRE

Fiig. 1-44 FFLJ00018 HH-RRas A, B, NIH3T3 EGFR, H-Ras (H-Ras CA), H-Ras (H-Ras DN), Myc FLJ00018

20 ng/ml, 15 EGF7.5% SDS-PAGE Myc FLJ00018

Anti-Myc

A B

22

(Fig. 1-5B, C) KN-93

EGF FLJ00018

MEK FLJ00018 SRE

CaMK MEK FLJ00018

FLJ00018 SRE CaMK

EGF FLJ00018 SRE EGF

MAPK FLJ00018 EGFR

p38-MAPK SB202190 JNK SP600125 30 EGF

FLJ00018

EGF FLJ00018 (Fig. 1-5D)

FLJ00018 SRE p38-MAPK JNK

SRE (Fig.

1-5E) 2 FLJ00018

A

B C

23

6. EGF FLJ00018

EGF FLJ00018 FLJ00018

EGFR FLJ00018

Fiig. 1--5 FLJ00018 MAPK A, D, NIH3T3 EGFR, Myc FLJ00018

CaMK KN-93 (10 M, A) MEK U0126 (10 M, A) p38MAPK SB202190 (10 M, D) JNK

SP600125 (10 M, D) 30 20 ng/ml, 15 EGF7.5% SDS-PAGE Myc FLJ00018

Anti-Myc B, C, E, NIH3T3 pSRE.L-luciferase, pRL-SV40 DNA, FLJ00018, EGFR

10 M KN-93 (B) 10 M U0126 (C) 10 M SB202190 (E) 10 M SP600125 (E)

1 20 ng/ml EGF 6dual-luciferase reporter assay system Mock 1.0

3 ± S.D.

D

E

D

24

Figure 1-6A FLJ00018 Figure 1-6B

FLJ00018 WT FLJ00018 P1 EGFR EGF

FLJ00018 P1.5, FLJ00018 P2, FLJ00018 P3,

FLJ00018 N EGFR

EGF FLJ00018

1 FLJ00018 616-970

A

B

C

25

P1.5, P2, P3, N EGF

Phos-tag Phos-tag P1.5, N

EGF (Fig. 1-6C)

FLJ00018 616-970 465-615, 965-1386

EGF P2, P3

P2 P3

311-464

77. EGF FLJ00018

Figure 1-5 FLJ00018 EGF MEK

Figure 1-6

FLJ00018 615-970

MEK ERK (44)

FLJ00018 680 680

(FLJ00018 T680A) EGF

(Fig. 1-7A) FLJ00018

T680A FLJ00018 WT EGF

Fiig. 1--6 EGF FLJ00018A, FLJ00018 WT, P1, P1.5, P2, P3, N FLJ00018

1-1386, 1-970, 1-615, 1-464, 1-310, 965-1386 B NIH3T3 EGFR, Myc FLJ00018

EGF (20 ng/ml) 15 7.5% SDS-PAGE Myc

FLJ00018 Anti-Myc , , FLJ00018 C, NIH3T3 EGFR, Myc FLJ00018

20 ng/ml EGF (15 ) 15 M Phos-tag AAL107, 30 M MnCl2 6%

SDS-PAGE Myc FLJ00018Anti-Myc , FLJ00018 ,

FLJ00018

26

FLJ00018 680 FLJ00018

HEK293 EGFR FLJ00018 WT FLJ00018 T680A

(Fig. 1-7B) Phos-tag phos-tag FLJ00018

WT FLJ00018 T680A EGF

(Fig. 1-7C) EGF ERK

FLJ00018 680 EGF

FLJ00018 T680A FLJ00018

680 EGF

FLJ00018 680 FLJ00018

SRE Figure 1-7D FLJ00018 T680A EGF

SRE FLJ00018 WT Rho

FLJ00018 T680A RhoGTPase

FLJ00018 T680A FLJ00018WT (Fig. 1-3B) Rac, Cdc42

RhoGTPase (Fig.

1-7E)

27

88. EGF FLJ00018

Fiig. 1--7 EGF FLJ000188A, B, NIH3T3 (A) HEK293 (B) EGFR, Myc FLJ00018

EGF (20 ng/ml) 15 7.5% SDS-PAGEMyc FLJ00018 Anti-Myc

C, HEK293 EGFR, Myc FLJ0001820 ng/ml EGF (15 )

13 M Phos-tag AAL107, 26 M MnCl2, 1.5% 3% SDS-PAGE Myc FLJ00018 Anti-Myc

D, E, NIH3T3 pSRE.L-luciferase, pRL-SV40 DNA, FLJ00018, EGFR, pF4A-RhoA T19N, pF4A-Rac T17N, pF4A-Cdc42 T17N

20 ng/ml, 6 EGFdual-luciferase reporter assay system Mock 1.0

3 S.D.

none

EGF

mAG mAG/EGFR mAG-018WT mAG-018 WT/EGFR mAG-018 T680A mAG-018 T680A/EGFR

B

C

D

A E G

F H J

I K

L

28

RhoGEF

(29,45) EGF FLJ00018

Neuro-2a FLJ00018, EGFR EGF (20 ng/ml,

24 ) FLJ00018

NIH3T3 HEK293 EGF FLJ00018 (

) Neuro-2a

(mAG) (32) mAG FLJ00018 (mAG-FLJ00018) EGFR

FLJ00018 WT EGFR EGF

(Fig. 1-8G, H) FLJ00018

mAG-FLJ00018 T680A EGFR FLJ00018 WT

EGF (Fig. 1-8K, L) FLJ00018

680

Figure 1-8M FLJ00018 WT

FFig.. 1--88 EEGF FFLJ00018 A-L, Neuro-2a mAG, mAG FLJ00018 EGFR

3 5% FBS20 ng/ml EGF 24

4% M, sample 10 50

Image J

M

29

EGFR FLJ00018 T680A EGFR

EGF

FLJ00018 680 EGF

30

4

FLJ00018 G G

RhoGTPase Rac1, Cdc42 (23)

FLJ00018 1AR EGFR transactivation

(Fig. 1-2) endothelin, thrombin, calbachol, LPA GPCR

EGFR transactivation GPCR EGFR

transactivation

ADAM/heparin-binding EGF (HB-EGF) EGFR LPA

LPA HB-EGF EGFR

phospholipase C 1 (PLC 1) PI3K/AKT (46)

2AR c-Src EGFR ERK PLC 1 PI3K/AKT

(47) AR EGFR

transactivation

Dakka 2AR Gs Gi

Gi G , Src Ras/MAPK

(48) Gs/Gi

Src EGFR/MAPK (49) GRK5

GRK6/ -arrestin/Src/EGFR transactivation

(40) AR EGFR transactivation Ras-MAPK

FLJ00018 AR

EGFR/Ras/MAPK


Vascular smooth muscle Thrombin protease-activated receptors 1

(PAR1) EGFR transactivation Thrombin EGFR

31

RhoA (50) EGFR transactivation

RhoGEF 2AR /Gi

Src (PDGFR)/PI3K

(39) 1AR EGFR - EGFR

EGFR transactivation

(42) FLJ00018, 1AR -ARKct

1AR SRE FLJ00018 T680A

FLJ00018 WT SRE


FLJ00018 EGFR (Fig.

1-3) EGF FLJ00018 Ras-MAPK (Fig. 1-4, 1-5)

FLJ00018 (Fig. 1-5) EGFR

RhoGEF

(27) RhoGEF Asef EGFR

Src Tyr 94

(28) Asef Rac1, Cdc42 RhoGEF PIX

FGFR Ras-ERK-Pak2 Ser 525 Thr 526

(29) PIX PC12

PIX EGFR

Src Tyr 442 (51)

RhoGEF Rac1, Cdc42 GEF

RhoA GEF GEF-H1 ERK1/2 Thr 678

(52) RhoGEF

RhoGEF GPCR EGFR

32

RhoGEF

Tiam1 TrkA TrkB

NGF TrkA Tiam1 Ras Rac

(53) Burridge EGF

RhoG RhoG Vav RhoGEF

RhoG RhoGEF PLEKHG6 (54)

RhoGEF Trio RhoG

(55) Ras/MAPK

FLJ00018 FLJ00018

FLJ00018

EGF FLJ00018 T680A

RhoGTPase 680

Rho

PIX

RhoGEF

EGF FLJ00018

FLJ00018

33

FFiig. 1--99 EEGFR ttransactivation FFLJ00018 1AR EGFR transactivation EGFR MAPK

FLJ00018 FLJ00018 MAPK ERK1/2 680FLJ00018 MAPK MEK

465-615 965-1386 /FLJ00018 RhoGTPase Cdc42, Rac1

1AR

transactivation

EGFR

Isoproterenol

H-Ras

MEK

MAPKUNKNOWN

cytoskeltal regulation

DH PH

T680

Cdc42 Rac activation

FLJ00018

UNKNOWN

34

2 Src

1

FLJ00018 EGFR Ras-MAPK

EGF FLJ00018

EGF FLJ00018

90%

%

(RTK) RTK

EGFR (Trk) (IGFR)

(FGFR) (PDGFR)

(VEGFR) (HGFR)

(FAK) Src FAK

paxillin p130Cas

FAK

(56-62) Src

Src, Yes, Fyn, Lyn, Lck, Blk, Hck, Fgr

1

Src homology 2 SH2

(63-66)

35

RhoGEF

Tiam Cool-1 Src

(51,67) Rac GEF Vav2 Src

(68) FLJ00018 Src

Src Src FLJ00018

FLJ00018

36

2

1.

pF1K ABL1, PIK3R3, EphA1, EphA3, EphA5, EphB2

DNA PCR

pF5A-CMV-neo-Flag pSUEamp-cSrc Millipore

pSUEamp-cSrcCA pSUEamp-c-Src PCR (Stratagene)

pFN21A-Myc FLJ00018

pFN21A-Myc-FLJ00018 WT PCR (Stratagene)

pSRE.L-luciferase reporter Stratagene pRL-SV40 Nippon Gene

Src PP2 Tocris ABL GNF-2 Sigma-Aldrich

2.

HEK293 37 , 5% CO2 10% FBS DMEM

Lipofectamine2000

(Life Technologies) SRE 0.4 g DNA

1 g DNA 2 g DNA

37 6-8 16-18

3. SH2

SH2 N Halo-tag/His SH2

DNA SH2 (Open

Biosystems ) PCR pFN18A

SH2 KRK Halo-tag SH2

Ni-NTA SH2 200-300 ng

HaloLink Protein Array slide (Promega)

37

0.05% IGEPAL CA630 (sigma) PBS FLJ00018 489

SH2 Myc

FLJ00018 WT FLJ00018 Y489F HEK293 10 l/well

SH2 SH2

Myc-FLJ00018 Myc

44.

PBS 1% (w/v) SDS / 20 mM Tris-HCl / 1 mM EDTA (pH 8.0)

1× SDS sample buffer

BCA protein assay kit (Thermo Scientific)

7.5% 10% SDS-PAGE

PVDF 2% TBS-T 10 Myc-

FLJ00018 1:2000 Anti-Myc

(Roche, 0.2 g/ml) 1:5000 HRP- IgG

(MBL) 1:2000

Anti-P-Tyr (PY20) (Santa Cruz Biotechnology, stock 100 g/ml)

1:5000 HRP- IgG

enzyme-linked chemiluminescence (GE Healthcare)

LAS-4000 mini image analyzer (GE)

5.

6 cm dish 300 l (50 mM

Tris-HCl, pH 7.5, 100 mM NaCl, 0.1 mM EDTA, 1 mM Na3VO4, 0.5% Nonidet P-40,

phosphase inhibitor, protease inhibitor) 13200 rpm, 10 min

1.0 g Anti-Myc 4 2

38

protein G-agarose 4 1

wash buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.1 mM EDTA, 1 mM Na3VO4,

0.1% Nonidet P-40, phosphase inhibitor solution, protease inhibitor) 3

sample buffer 7.5% 10

SDS-PAGE

39

3

1. FLJ00018 cSrcCA

FLJ00018 Src HEK293 cSrc

cSrcCA FLJ00018

FLJ00018 SrcCA

(Fig. 2-1)

2. cSrcCA FLJ00018

Src FLJ00018 FLJ00018

cSrcCA

FLJ00018 Figure 2-2A

FLJ00018 WT FLJ00018 P2 FLJ00018 P1.5 cSrcCA

FLJ00018 (Fig. 2-2B) Src

FLJ00018 1 FLJ00018 465-615

FLJ00018 465-615

489 510 2 cSrc

489 510

Fiig. 2--1 cSrcCA FLJ00018HEK293 Myc FLJ00018 cSrc cSrcCA

7.5% SDS-PAGEMyc FLJ00018 Anti-Myc

FLJ00018 Anti-p-Tyr

40

FLJ00018 Y489F, FLJ00018 Y510F cSrcCA

Figure 2-2C

FLJ00018 Y510F FLJ00018 Y489F

cSrcCA FLJ00018

489

FFiig. 22--22 ccSrcCA FFLJ00018A, FLJ00018 WT, P1, P1.5, P2 FLJ00018 1-1386, 1-970, 1-615, 1-464 B, C, HEK293 Myc FLJ00018WT SrcCA

7.5% SDS-PAGEMyc FLJ00018 Anti-Myc FLJ00018

Anti-p-Tyr

41

33. FLJ00018 489

SH2

(69,70) SH2

121 38 (63) FLJ00018

489 SH2

SH2 SH2

FLJ00018 WT FLJ00018 Y489F cSrcCA SH2

PVDF FLJ00018 FLJ00018

Y489F ABL1 PIK3R3 (Fig. 2-3) ABL1 Src

PIK3R3 (PI)

PI-3

Fiig. 2--3 FLJ0001850 SH2 Halo HaloLink

(N) (C) 2 SH2N C SH2

WT, Y489F SH2 WT SH2

42

44. FLJ00018 ABL1 PIK3R3

ABL1 PIK3R3 FLJ00018

ABL1 WT PIK3R3 WT

cSrcCA FLJ00018 WT Y489F ABL1

FLJ00018 WT FLJ00018 Y489F ABL1 (Fig.

2-4A) FLJ00018 Y489F PIK3R3 FLJ00018 WT

PIK3R3 (Fig. 2-4B) PI3KR3 FLJ00018 489

ABL1 SH2

FLJ00018 489 SH2

FLJ00018

5. ABL1 cSrcCA FLJ00018

ABL1 Src ABL1 FLJ00018

FLJ00018 ABL1 Src

489 (Fig. 2-5A) ABL1 Src

(71,72) Src

PP2 ABL1 GNF-2 Figure 2-5B

Fiig. 2--4 PIK3R3 ABL1 FLJ00018A, B, HEK293 Src Myc FLJ00018WT Y489F Flag- ABL1

PIK3R3 MycFLJ00018 7.5% SDS-PAGE

Myc FLJ00018 Anti-Myc Flag ABL1PIK3R3 Anti-Flag

A B

43

ABL1 FLJ00018 GNF-2 FLJ00018

cSrcCA FLJ00018 GNF-2

FLJ00018 FLJ00018 cSrcCA

PP2 ABL1 FLJ00018

PP2 FLJ00018

(Fig. 2-5C) ABL1 Src FLJ00018

B C

66. EphB2/Src FLJ00018

Asef Cool-1 RhoGEF EGFR

(28,51) FLJ00018 1 EGFR

B C

Fiig. 2--5 cSrcCA ABL1 FLJ00018A, B, C, HEK293 Myc FLJ00018WT Y489F cSrcCAABL1

GNF2 (10 M, B) PP2 (10 M, C) 167.5% SDS-PAGE Myc FLJ00018Anti-Myc FLJ00018 Anti-p-Tyr

44

Src

(Eph) Eph FLJ00018

Eph FLJ00018 HEK293

FLJ00018 Figure 2-6A

EphB2 FLJ00018 FLJ00018

EphB2 Src FLJ00018

FLJ00018 Y489F EphB2 FLJ00018

Y489F EphB2 (Fig. 2-6B)

FLJ00018 EphB2 Src

ABL1 489 EphB2

FLJ00018 Src ABL1 PP2

GNF-2 FLJ00018 EphB2 ABL1

GNF2 EphB2 FLJ00018

(Fig. 2-6C) Src PP2

EphB2 FLJ00018 (Fig. 2-6D)

EphB2 FLJ00018 489 Src

A

45

FFiig. 22--66 EphB2/cSrc FFLJ00018 A, HEK293 Myc FLJ00018 WT Eph

7.5% SDS-PAGEMyc FLJ00018 Anti-Myc

FLJ00018 Anti-p-Tyr B, C, D, HEK293 Myc FLJ00018 WT Y489F EphB2

GNF2 (10 M, C) PP2 (10 M, D) 167.5% SDS-PAGE Myc FLJ00018

Anti-Myc FLJ00018 Anti-p-Tyr

46

4

HEK293 FLJ00018 EphB2/Src

489 (Fig. 2-2)

SH2

SH2 FLJ00018 ABL1

PIK3R3 (Fig. 2-3) FLJ00018

PIK3R3

(Fig. 2-4)

RhoGEF Src Asef EGF

Src 94

(28) RhoGEF Vav1 Cool-1 Src

(51,73) RacGEF Tiam1

Src 389 (67) Vav

BCR-ABL (74) BCR-ABL ABL 9

BCR 22

Ras-GEF Sos-1 RTK ABL

Rac GEF (75)

RhoGEF 1 FLJ00018 Src ABL

ABL1 PDGF Src

(71,72) Src 2AR G

Src PDGF PIK3 EphB2 611

EphB2 Src (76)

Src ABL

FLL00018 Src FLJ00018

47

EphB2 Src

PDGFR

EGFR FLJ00018

EphB2/cSrc FLJ00018 ABL FLJ00018

ABL FLJ00018

FLJ00018 EphB2/Src FLJ00018

PIK3R3 EphB2

Src PIK3R3

FLJ00018

FFig. 2--77 EEphB2/Src FFLJ00018 AABL1 FFLJ00018 Eph cSrc FLJ00018 489

ABL1 ABL1FLJ00018 PIK3R3 SH2

48

3 FLJ00018

1

1 2 FLJ00018 FLJ00018

EGF EphB2/Src

FLJ00018 RhoGEF

Vav1 Vav1

(77)

RhoGEF PDZ-RhoGEF, LARG, p115RhoGEF GEF

DH PH N RGS

PDZ-RhoGEF LARG RGS N PDZ

GPCR G G 12/13

p115-RhoGEF RhoGEF RGS

(14,15) GPCR LARG PDZ-RhoGEF

PDZ GPCR Plexin B1

(78-82)

FLJ00018 1386

FLJ00018 GEF

FLJ00018

FLJ00018 DH, PH 1-465

466-965 966-1386 3

two-hybrid

49

FLJ00018 3 DH, PH N 1-465

two-hybrid

FLJ00018 1-465

1

50

2

1.

pEGFP- -actin Clontech -actin -actin cDNA PCR

pF5A-CMV-neo FLJ00018

pFN21A-Myc-FLJ00018 WT PCR (Stratagene)

pSRE.L-luciferase reporter Stratagene pRL-SV40 Nippon Gene

pF5A-CMV-neo-G 1 pF5A-CMV-neo-G 2 1

pCDNA3.1-Rac1 Missouri S&T cDNA Resource Center

RacG15A PCR

pGEX-4T1 phosphatase protease Roche

PLEKHG2 Bethyl Laboratories Flag

Myc Wako HRP GST GE

2. two-hybrid

two-hybrid Matchmaker Gold two-hybrid (Clontech)

bait FLJ00018 1-465

Gal4 DNA pGBKT7- beit

pGBKT7-FLJ00018 1-465 Y187 prey pGADT7

cDNA-Gal4 DNA

(Clontech) Y2H Gold Y187

Y2H Gold SD

4 SD

pGBKT7-FLJ00018 1-465 Y2H Gold

51

3.

HEK293 37 , 5% CO2 10% FBS DMEM

NIH3T3 37 , 5% CO2 10% CS DMEM

LipofetAMINE Lipofectamine2000


1 g DNA pull-down 2 g

DNA 37 6-8

16-18


24 pRL-SV40

pSRE.L-luciferase

PBS 1

13200 rpm, 10 min dual luciferase

reporter assay system (Promega)

±S.D.

5.




1.0 g Anti-Myc 4 2

protein G-agarose 4 1

wash buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.1 mM EDTA, 1 mM Na3VO4,

0.1% Nonidet P-40, phosphase inhibitor solution, protease inhibitor) 3

52

sample buffer 7.5% 10

SDS-PAGE

6. Pull-down

FLJ00018 DH5 GST-Rac G15A

(83) 6 cm dish HEK293 300 l

(50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.1 mM EDTA, 1 mM Na3VO4, 0.5%

Nonidet P-40, 10% Glycerol, phosphase inhibitor, protease inhibitor)

13200 rpm, 10 min

4B GST-Rac G15A 4 1

wash buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.1 mM EDTA, 1 mM

Na3VO4, 0.1% Nonidet P-40,10% Glycerol, phosphase inhibitor solution, protease inhibitor)

3 sample buffer

7.5% 10 SDS-PAGE

7.

PBS 30 4%

0.1% Triton-X PBS 4

10% goat serum 1 1

IgG-Alexa-Flour488 Alexa-Flour568 Phalloidin

Perma Fluor

(BZ-9000; KEYENCE)

53

3

1. FLJ00018

FLJ00018 FLJ00018 GEF DH PH

1-465 two-hybrid bait

FLJ00018 1-465 Y187 cDNA

FLJ00018 Y2H Gold

9.0 106 SD

1424

SD

971 285

FLJ00018 1-465 Y2H Gold

121

Figure 3-1 11-2

11-2

bait 11-2

Fig. 3--1 FLLJ00018 1-4465aa (Bait) 11-2 (Prey)

pGBKT7-FLJ00018 1-465aapGBKT7-DNA-BD pGADT7-11-2

Y2H Gold(DDO/X);

SD(QDO/X/A);

SD

54

GenBank

-

22. FLJ00018 - -

6

2

- - N 1, 2, 3 9

(84) two-hybrid - 257-376

- 257-376 - (Fig. 3-2A)

Flag- - - Myc FLJ00018

HEK293

Figure 3-2B FLJ00018 WT -

- FLJ00018 - -

two-hyabrid FLJ00018 1-465

- -

(Fig. 3-2C)

- FLJ00018

FLJ00018 WT FLJ00018 P2 -

FLJ00018 -

(Fig. 3-2D)

55

33. - FLJ00018

FLJ00018 two-hybrid 1-465

FLJ00018 449-1386 FLJ00018 NT

(Fig 3-3A) - Figure 3-3B

FLJ00018 NT - 1-465

Fiig. 3--2 FLJ00018A, FLJ00018 DH, dbl ; PH, pleckstrin

; Y2H, yeast two-hybrid prey B-C, HEK293 Myc FLJ00018 WT (B) FLJ00018 P2 (C) Flag-

- -Myc FLJ00018 SDS-PAGE

Myc FLJ00018 Anti-Myc FlagAnti-Flag

D, HEK293 Myc FLJ00018WT FLJ00018 P2Myc FLJ00018

SDS-PAGE Myc FLJ00018Anti-Myc Anti- - (*)

Myc-

56

FLJ00018

FLJ00018 2

FLJ00018 1-465

1-465aa N C (Fig. 3-4A)

FLJ00018 P2 (Bait) N P2 N1

P2 N2 P2 N3 -

Bait F018 P2 P2 N1 P2 N2 -

P2 N3 - (Fig. 3-4B) bait

P2 C P2 C1, P2 C2, P2 C3 PH

FFiig. 33--33 FFLJ00018 NNTA, FLJ00018 NT FLJ00018 NT FLJ00018 449-1386

B, HEK293 Myc FLJ00018 WT FLJ00018 P2, FLJ00018 NTFlag- - Myc

FLJ00018 SDS-PAGE MycFLJ00018 Anti-Myc Flag

Anti-Flag (*) Myc-

57

- P2 C1, P2 C2 P2 C3

- PH (Fig. 3-4C)

FLJ00018 P2 FLJ00018 DH 150

283 -Actin

44. - - FLJ00018 SRE

FLJ00018 FLJ00018

- - FLJ00018 WT P2

Fiig. 3--4 FLJ00018 P2A, FLJ00018 P2 P2, P2 N1, P2 N2, P2 N3, P2 C1, P2 C2, P2 C3, PH

FLJ00018 1-464, 58-464, 150-464, 283-464, 1-441, 1-355, 1-310 283-441

B, C, Myc FLJ00018 Flag- - HEK293Myc FLJ00018

SDS-PAGE Myc FLJ00018Anti-Myc Flag Anti-Flag

(*) Myc-

58

FLJ00018 SRE

-

FLJ00018 FLJ00018 SRE -

- - FLJ00018 SRE -

FLJ00018 G G

G FLJ00018 SRE

FLJ00018 G

FLJ00018 SRE

(Fig. 3-5)

59

- FLJ00018 DH5

GST-Rac G15A pull-down FLJ00018 RhoGTPase GEF

Rac G15A Rac

RhoGEF RhoGEF (83) Figure 3-6

FLJ00018 FLJ00018 -

GST-Rac G15A FLJ00018

FLJ00018 FLJ00018

FFiig. 33--55 FFLJ00018 SSRE A, C, HEK293 pSRE.L-luciferase, pRL-SV40 DNA, FLJ00018 WT (A), FLJ00018 P2 (C), G , G , - -

dual-luciferase reporter assay systemMock 1.0 3 ±

S.D. B, D, HEK293 , Myc FLJ00018 WT (B) FLJ00018 P2 (D) Flag

- - G , G7.5% SDS-PAGE Myc

FLJ00018 Anti-Myc FLJ00018Anti-PLEKHG2 Flag - - Anti-Flag

- Anti- - GAnti-G

FFiig. 33--66 FFLJ00018 HEK293 , Myc FLJ00018 WTFlag -

GST-Rac G15ASDS-PAGE

60

55. - FLJ00018 G

FLJ00018 SRE FLJ00018

G FLJ00018 G -

HEK293 FLJ00018

G - Figure 3-7 FLJ00018 G

- - G

FLJ00018

6. FLJ00018

FLJ00018 NIH3T3

FLJ00018 G (Fig. 3-8A, B)

FLJ00018 FLJ00018 G

FLJ00018 F- (Fig. 3-8C)

FLJ00018 FLJ00018 F-

FLJ00018 F-

Fiig. 3--7 FLJ00018 GG -

Myc FLJ00018 Flag-- G G HEK293

Myc FLJ00018SDS-PAGE

62

Fiig. 3--8 FLJ00018 A, NIH3T3 Myc FLJ00018 WT G , G

SDS-PAGE Myc FLJ00018 Anti-Myc G

Anti-G B, C, NIH3T3 Myc FLJ00018 WT G , G

G (B)FLJ00018 (C) F- , 20 m

63

4

G RhoGEF FLJ00018 G

FLJ00018 2

two-hybrid FLJ00018 1

150-283 FLJ00018 N

G FLJ00018

FLJ00018 G

FLJ00018 - FLJ00018

(Fig. 3-9)

FLJ00018

- - FLJ00018

- -

(84,85) FLJ00018 - -

- - FLJ00018

in vitro FLJ00018

- -

FLJ00018

myosin -Pix Dbl RhoGEF

myosin DH, PH

RhoGEF (86) -Pix-bL N

calponin homology

(87) RhoGEF

PDZ-RhoGEF PDZ-RhoGEF

PDZ-RhoGEF (88,89) RhoGEF

RhoGEF

64

FLJ00018

PDZ-RhoGEF

RhoGEF L/IIxxFE

(89) FLJ00018 662-667 IxxxFE

2 C

150-283 L/IIxxFE

FLJ00018 150-283

FLJ00018 RhoGEF PLEKHG1 PLEKHG3

DH (60% )

FFig. 3--99 FFLJ00018 GPCR G FLJ00018 FLJ00018

F- FLJ00018 150-283aaFLJ00018

65

4 FLJ00018 Four and a half LIM 1 (FHL1)

1

3 FLJ00018 1-465

two-hybrid 24

Genbank (>80%)

two-hybrid 24 Four and

a half LIM domain 1 (FHL1)

FHL myopathy

(90,91) FHL3

- FHL1

(92,93)

FHL1

(94) FHL1

FHL2 FHL1 mRNA FHL1

FHL1, FHL2 mRNA

(95) FHL1 Src

Crk-associated substrate (Cas) Src

(96)

FHL1 FHL1

two-hybrid prey cDNA FLJ00018

FHL1 FHL1

FLJ00018 FHL1

66

FHL1 FLJ00018

67

2

1.

FHL1A FHL1B ORF cDNA PCR

pF5A-Flag FLJ00018 pSRE.L-luciferase reporter

pRL-SV40 pF5A-CMV-neo-G 1 pF5A-CMV-neo-G 2 1 3

2.

HEK293 Neuro-2a 37 , 5% CO2 10% FBS DMEM

Lipofectamine2000


1 g DNA pull-down 2 g

DNA 37 6-8

16-18


24 pRL-SV40

pSRE.L-luciferase

PBS 1

13200 rpm, 10 min dual luciferase

reporter assay system (Promega)

±S.D.

4.



68


1.0 g Anti-Myc 1.0 g Anti-Flag

4 2 protein G-agarose 4 1

wash buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.1

mM EDTA, 1 mM Na3VO4, 0.1% Nonidet P-40, phosphase inhibitor solution, protease

inhibitor) 3 sample buffer

SDS-PAGE

55.

pFN21A-mAG-FLJ00018

PBS 30 4%

0.1% Triton-X PBS 4 10% goat serum 1

Anti-Flag 1 IgG-Alexa-Flour568

Perma Fluor

(BZ-9000; KEYENCE)

69

3

1. FLJ00018 Four and a half LIM domain1 (FHL1)

3 FLJ00018 1-465 two-hybrid

121 24 Genbank

(>80%) 3 24 1

FLJ00018

5-1

(Fig. 4-1) 5-1

bait 5-1

Four and a half LIM

domain1 (FHL1)

2. FLJ00018 FHL1

FHL1 LIM

LIM CX2CX16-23HX2CX2CX2CX16-21CX(C,H,D) 2

Zinc Zinc FHL FHL1-4, ACT 5

FHL1 FHL1A, 1B, 1C 3

Fig. 4--1 FLLJ00018 1-4465aa (Bait) 5-1 (Prey)

pGBKT7-FLJ00018 1-465aapGBKT7-DNA-BD pGADT7-5-1

Y2H Gold(DDO/X);

SD(QDO/X/A);

SD

70

(97) FHL1A 4 LIM FHL1B

3 LIM C (NLS)

two-hybrid FHL1A FHL1B LIM2 3

(Fig. 3-2A) Flag- FHL1A FHL1B

Myc FLJ00018 HEK293

Figure 3-2B

FLJ00018 WT FHL1A FHL1B FLJ00018

FHL1A FHL1B

FLJ00018 FHL1B FHL1A

FHL1B NLS (98)

A

B

FFiig. 44--22 FFLJ00018 FFHL1A, FHL1 prey two-hybrid FLJ00018

B, HEK293 Myc FLJ00018WT Flag- FHL1A FHL1B

MycFLJ00018 SDS-PAGE Myc

FLJ00018 Anti-Myc Flag FHL1Anti-Flag

71

33. FHL1 FLJ00018

FHL1 two-hyabrid

FLJ00018 1-465

FLJ00018 P2 FLJ00018 NT FHL1 HEK293

two-hybrid FLJ00018 1-465 P2

FLJ00018 NT FHL1 (Fig. 4-3A, B)

FLJ00018 2 FHL1

A B

4. FHL1 FLJ00018 P2

FLJ00018 1-465

1-465aa N C ( 3 Fig. 3-4A) FHL1A

bait P2 N

P2 N1 P2 N2 P2 N3 FHL1A

bait F018 P2 P2 N1 FHL1A

P2 N2 P2 N3 FHL1A

Fiig. 4--3 FLJ00018 NT FHL1A, B HEK293 Myc FLJ00018WT FLJ00018 P2, FLJ00018 NTFlag- FHL1A (A) FHL1B (B)

Myc FLJ00018 SDS-PAGEMyc FLJ00018 Anti-Myc Flag

FHL1 Anti-Flag

72

(Fig. 4-4A) bait P2 C P2 C1, P2 C2,

P2 C3 PH FHL1A P2 C1, P2

C2 P2 C3 FHL1A PH

(Fig. 4-4B) FHL1 FLJ00018 P2 -

FLJ00018 N 58 150

A

B

Fiig. 4--4 FLJ00018 P2 FHL1A, B, Myc FLJ00018 Flag FHL1A HEK293

Myc FLJ00018SDS-PAGE Myc FLJ00018

Anti-Myc Flag FHL1 Anti-Flag

73

55. FHL1 FLJ00018 SRE

FLJ00018

FHL1 FHL1 FLJ00018

FHL1A FHL1B FLJ00018 WT P2

FLJ00018 SRE

Figure 4-5A FHL1A FLJ00018 FLJ00018

SRE FHL1B FLJ00018 FLJ00018

SRE (Fig.

4-5B)

A B

Fiig. 4--5 FHL1 FLJ00018 SRE A, HEK293 pSRE.L-luciferase, pRL-SV40 DNA, FLJ00018 WT, FLJ00018 P2, FHL1A 1B

dual-luciferase reporter assay system Mock 1.03 ± S.D.

B, HEK293 , Myc FLJ00018 WT FLJ00018 P2, FlagFHL1A FHL1B

SDS-PAGE Myc FLJ00018Anti-Myc Flag FHL1 Anti-Flag

74

FLJ00018 P2 FHL1 FLJ00018 G G

G FLJ00018 SRE

FHL1 FHL1A G

FLJ00018 (Fig. 4-6A, D)

(Fig. 4-6B-C,

E-F) FHL1A FLJ00018 SRE

FLJ00018

A D

B E

C F

75

6. FHL1 FLJ00018 G

FHL1A FLJ00018 SRE

FLJ00018 G

FHL1A FLJ00018 G

FLJ00018 G FHL1A HEK293 FLJ00018

G FHL1A Figure 4-7A FLJ00018

G FHL1A G FLJ00018

FHL1A FLJ00018

FHL1A G (Fig. 4-7B)

FLJ00018 FHL1A, G

FHL1A G FLJ00018

FHL1A, G FLJ00018 FHL1A G

HEK293

Figure 4-7C FHL1A G FHL1 FLJ00018, G

FLJ00018 G FHL1A

Fiig. 4--6 FHL1 G FFLJ00018 SSRE A, D HEK293 pSRE.L-luciferase, pRL-SV40 DNA, FLJ00018 WT (A), FLJ00018 P2 (D), G , G , FHL1A FHL1B

dual-luciferase reporter assay systemMock 1.0 3 ± S.D.

B-C, E-F HEK293 , Myc FLJ00018 WT (B, C) FLJ00018 P2 (E, F)Flag FHL1A 1B G , G

SDS-PAGE Myc FLJ00018 Anti-Myc Flag FHL1

Anti-Flag G Anti-G

76

A B

C

7. FHL1 FLJ00018 SRE

FHL1A FLJ00018 FLJ00018 SRE

FHL1 G , FLJ00018

3 - FLJ00018 FLJ00018

- G FLJ00018 -

FLJ00018 FHL1

Fiig. 4--7 FHL1 FLJ00018 G A, B, Myc FLJ00018 Flag- FHL1A, G G HEK293


C, Myc FLJ00018 Flag FHL1A, G G HEK293Flag FHL1A

SDS-PAGE

77

FLJ00018 - FLJ00018, FHL1 -

HEK293 SRE FHL1 FLJ00018

SRE (Fig. 4-8A)

(Fig. 4-8B) FHL1 FLJ00018 -

FLJ00018

A B

8. FLJ00018 FHL1

FLJ00018 FHL1 Neuro-2a FLJ00018

FHL1, G Neuro-2a HEK293

FHL1A FHL1B (Fig. 4-9A, D) FLJ00018

SRE FHL1A FLJ00018

(Fig. 4- 9B, E) Neuro-2a FHL1 HEK293

FLJ00018

Fiig. 4--8 - FHL1 FLJ00018 SRE A, HEK293 pSRE.L-luciferase, pRL-SV40 DNA, FLJ00018 WT, FHL1A -


B, HEK293 Myc FLJ00018 WT, Flag FHL1A -

SDS-PAGE Myc FLJ00018Anti-Myc Flag FHL1 - Anti-Flag

78

B E

A

C

D

F

FFiig. 44--99 Neuro--22a FFHL1 FFLJ00018 FFLJ00018 A, D, Myc FLJ00018 Flag FHL1A FHL1B Neuro-2a


B, E, Neuro-2a pSRE.L-luciferase, pRL-SV40 DNA, FLJ00018 WT, FHL1AFHL1B, G , G


C, F, Neuro-2a Myc FLJ00018 WT, Flag FHL1AFHL1B, G , G

SDS-PAGE Myc FLJ00018Anti-Myc Flag FHL1 Anti-Flag

G Anti-G

79

Neuro-2a FLJ00018 FHL1A, FHL1B

FLJ00018 FHL1B

FHL1B FLJ00018

FHL1B FLJ00018 G

FHL1B FLJ00018 (Fig. 4-10B)

FHL1A FLJ00018 FHL1A FLJ00018

FHL1A

FLJ00018 G

(Fig. 4-10C) FLJ00018 G

(Fig. 4-10A) FLJ00018 FHL1A

A

80

B

C

81

Fiig. 4--10 FLJ00018 FHL1A A-C, Neuro-2a mAG FLJ00018 WT Flag-FHL1A FHL1B, G , G

Anti-Flag FHL1, 40 m

82

4

3 FLJ00018 1-465

two-hybrid 24

Genbank (>80%)

two-hybrid 24

FHL1

FHL1 Zinc LIM 4

two-hybrid FLJ00018 FHL1 N

2 LIM 3 LIM

FHL1 FHL1A, FHL1B FHL1C FHL1A FHL1B

FHL1A FHL1B

FHL1A (Fig.

4-2B) FHL1B NLS

(98) FHL1B

(Fig. 4-10B, C)

FHL1B G2

2A (PP2AC )

(99) FHL1B

FLJ00018

FLJ00018 N 58-150 FHL1A

FLJ00018 (Fig. 4-4, 4-5) FLJ00018

1-465 FHL1A

FLJ00018 DH N FLJ00018

FLJ00018 FHL1A FLJ00018

G FLJ00018, FHL1A, G

83

3 (Fig. 4-7) FHL1A

FLJ00018 G SRE

Neuro-2a FLJ00018 FHL1A

G FLJ00018

FHL1A FHL1A FLJ00018 G

FLJ00018

FHL ACT, FHL2, FHL3 (AR)

(100-102) FHL2 AR

Rho FHL2 (103)

FHL2

(104,105) FHL2 ERK2

ELK-1, GATA4 (106) FHL

FLJ00018 FHL1 FLJ00018

G

two-hybrid prey cDNA

FLJ00018 FHL1 FLJ00018

FLJ00018

84

Fiig. 4--11 FHL1 FLJ00018 FLJ00018 FHL1

FHL1 FLJ00018 G

85

GPCR

G , G , G 3 G GPCR

G

Ras RhoA Small GTPase

RhoGTPase

RhoGTPase RhoGEF G

RhoGTPase G G

RhoGEF G

RhoGEF PLEKHG2/FLJ00018 RhoGTPase Rac1 Cdc42

FLJ00018

G (1) (2)

(1) FLJ00018

FLJ00018 Gs GPCR 1AR EGFR transactivation

FLJ00018 EGFR

FLJ00018

EGF FLJ00018

680

86

FLJ00018

FLJ00018 EGF

RhoGEF

Src

FLJ00018 FLJ00018 489

Src SH2

FLJ00018 SH2

PIK3R3 ABL1

(2) FLJ00018

FLJ00018 DH

PH FLJ00018 1-465 bait cDNA Prey

two-hybrid FHL1 -, -

FLJ00018 -, -

FHL1 -, - FLJ00018 DH

150-283 aa FHL1 FLJ00018 58-150 aa

FHL1 -, - FLJ00018

-, - FHL1

FHL1 G

FLJ00018 58-150 aa

G FLJ00018

GPCR

GPCR

87

RhoGTPase

GPCR RhoGTPase

RhoGEF

FLJ00018 lymphoma

FLJ00018

FLJ00018

Fiig. 1 FFLJ00018 G FLJ00018 (1) EGFR

680 (2) EphB2/Src 489 PIK3R3

(3) FLJ00018(4) FHL1 FLJ00018

88

DNA

89

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spreading and stress fiber disassembly. J Biol Chem 2278, 24139-24152 94. Lee, S. M., Li, H. Y., Ng, E. K., Or, S. M., Chan, K. K., Kotaka, M., Chim, S. S., Tsui, S. K.,

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96. Shen, Y., Jia, Z., Nagele, R. G., Ichikawa, H., and Goldberg, G. S. (2006) SRC uses Cas to

suppress Fhl1 in order to promote nonanchored growth and migration of tumor cells. Cancer Res 666, 1543-1552

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domain protein FHL1B interacts with PP2A catalytic beta subunit--a novel cell cycle

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CREM and CREB by the LIM-only protein ACT. Nature 3398, 165-169 101. Fimia, G. M., De Cesare, D., and Sassone-Corsi, P. (2000) A family of LIM-only transcriptional

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98

ABL1 Abelson tyrosine kinase

APC Adenomatous polyposis coli

AR Androgen receptor

BCA Bovine serum albumin

Cas Crk-associated substrate

CaMK Calmodulin-Dependent Protein Kinase

cDNA Complementary DNA

CS Calf serum

CZH CDM-zizimin homology

DH domain Dbl homology domain

DHR2 Dock homology region 2

DMEM Dulbecco's Modified Eagle Medium

EGF Epidermal growth factor

EGFR Epidermal growth factor receptor

Eph Ephrin receptor

ERK Extracellular signal-regulated kinase

FAK Focal adhesion kinase

FBS Fetal Bovine Serum

FGFR Fibroblast growth factor receptor

FHL1 Four and a half LIM domain 1

GAP GTPase activation protein

GDI GDP dissociation inhibitor

GDP Guanosine diphosphate

GEF guanine nucleotide exchange factor

GPCR heterotrimeric G-protein coupled receptor

99

G-protein Guanosine triphosphate-binding protein

GTP Guanosine triphosphate

HB-EGF Heparin-binding EGF

HGFR Hepatocyte growth factor receptor

HRP Horseradish peroxidase

IGFR Insulin-like growth factor receptor

LARG leukemia-associated RhoGEF

LPA Lysophosphatidic acid

MAPK Mitogen-activated Protein Kinase

MEK MAP kinase kinase

NLS Nuclear localization signal

ORF Open reading flame

PAR1 Protease-activated receptors 1

PBS Phosphate buffered saline

PCR Polymerase chain reaction

PDGFR Platelet-derived growth factor receptor

PDZ domain PSD-95/Dlg/Zo-1 domain

PH domain Pleckstrin homology domain

PIK3 Phosphatidylinositol-3 kinase

PIK3R3 Phosphatidylinositol-3 kinase regulatory subunit3

PLC 1 Phospholipase C 1

PLEKHG2 pleckstrin homology domain containing, family G

PP2AC Protein phosphatase 2A catalytic subunit C

RGS domain Regulator of G-protein signaling domain

RhoGEF Rho family specific GEF

RhoGTPase Rho family specific small GTPase

100

RTK Receptor tyrosine kinase

SDS Sodium dodecyl sulfate

SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

SH2 Src homology 2

SRE Serum response element

TBS Tris buffered saline

VEGFR Vascular Endothelial Growth Factor Receptor

1AR 1-Adrenergic receptor

-ARKct C-terminal of -adrenergic receptor kinase

101

1. Katsuya Sato, Hiroaki Handa, Masashi Kimura, Yukio Okano, Hitoshi Nagaoka, Takahiro Nagase, Tsuyoshi Sugiyama, Yukio Kitade, and Hiroshi Ueda (2013) Identification of a Rho family specific guanine nucleotide exchange factor, FLJ00018, as a novel actin-binding protein. Cellular Signalling 225, 41-49

2. Katsuya Sato, Takahiro Suzuki, Yoshihiro Yamaguchi, Yukio Kitade, Takahiro

Nagase, and Hiroshi Ueda (2014) PLEKHG2/FLJ00018, a Rho family-specific guanine nucleotide exchange factor, is tyrosine phosphorylated via the EphB2/cSrc signaling pathway. Cellular Signalling 226, 691-696

3. Katsuya Sato, Tsuyoshi Sugiyama, Takahiro Nagase, Yukio Kitade, and Hiroshi Ueda, Threonine 680 phosphorylation of FLJ00018/PLEKHG2, a Rho family-specific guanine nucleotide exchange factor, by epidermal growth factor receptor signaling regulates cell

morphology of Neuro-2a cells. Journal of Biological Chemistry, in press

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