FLJ00018
ࡢάᛶࡢࣞ࣋ࣝࡣࠊ⭠⳦DH5Dᰴ࡛ᇵ㣴ࡋࠊ⢭〇ࡉࢀࡓ GST-Rac G15A
ࢆ⏝࠸࡚ホ౯ࡋࡓ (83)ࠋ6 cm dish୍㐣ᛶࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡉࢀࡓ
HEK293
⣽⬊ࢆ300 Pl
ࡢ⣽⬊⁐ゎᾮ (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.1 mM EDTA, 1 mM Na3VO
4, 0.5%
Nonidet P-40, 10% Glycerol, phosphase inhibitor, protease inhibitor)
୰࡛⁐ゎࡋࡓࠋ⁐ゎᾮࢆ
13200 rpm, 10 min
࡛㐲ᚰᚋࠊୖΎࡢ୍㒊ࢆศࡅࡾࠊṧࡾࡢୖΎࢢࣝࢱࢳ࢜ࣥࢭࣇ࣮ࣟࢫ
4B
ࣅ࣮ࢬࡀ⼥ྜࡋࡓGST-Rac G15A
ࢆຍ࠼ࠊ4Υ࡛ 1
㛫࣮ࣟࢸ࣮ࢩࣙࣥࢆ⾜ࡗࡓࠋࡑࡢ ᚋࣅ࣮ࢬࢆwash buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.1 mM EDTA, 1 mM Na
3VO
4, 0.1% Nonidet P-40,10% Glycerol, phosphase inhibitor solution, protease inhibitor)
࡛
3
ᅇὙίᚋࠊࣅ࣮ࢬ⤖ྜࡋࡓ⺮ⓑ㉁ࢆsample buffer
୰࡛⁐ฟࡋࡓࠋ୍ᐃ㔞ࡢචỿ㝆≀ࡣ7.5%ࡶࡋࡃࡣ 10㸣 SDS-PAGE
࡛ศ㞳ࡉࢀࠊ⺮ⓑ㉁ࡣ࣒ࣀࣈࣟࢵࢺἲࢆ⏝࠸᳨࡚ฟࡋࡓࠋ7.
⣽⬊චᰁⰍ⣽⬊ᙧែࡢほᐹ࢝ࣂ࣮࢞ࣛࢫୖ୍࡛㐣ᛶᙉไⓎ⌧ࡉࡏࡓ⣽⬊ࢆị෭
PBS
୍࡛ᅇὙίᚋࠊ30
ศ㛫ࠊ4%
ࣃ࣒ࣛ࣍ࣝࣝࢹࣄࢻᅛᐃࡉࡏࡓࠋᅛᐃࡋࡓ⣽⬊
0.1% Triton-X
ฎ⌮ࢆ⾜࠸ࠊPBS
࡛4
ᅇὙίᚋࠊ10% goat serum
࡛1
㛫ࣈࣟࢵ࢟ࣥࢢࡋࡓࠋࡑࡢᚋࠊྛᅗ♧ࡋࡓ୍ḟᢠయ࡛1
㛫ࣛ࣋ࣝࡋࠊ ࡉࡽࠊᢠ࣐࢘ࢫIgG-Alexa-Flour488
ཬࡧAlexa-Flour568 Phalloidin
࡛ᰁⰍࡋࡓࠋᰁⰍࡋࡓ࢝ࣂ࣮࢞ࣛࢫࡣࠊPerma Fluor ࡛ฎ⌮ࡉࡏࠊ⺯ග࣓࣮ࢪࢆ⺯ග㢧ᚤ㙾ࡼࡾほᐹࡋࡓ
(BZ-9000; KEYENCE)ࠋ
53
➨
➨ 3 ⠇ ⤖ᯝ
1. FLJ00018
┦స⏝ศᏊࡋ࡚ࡢ㠀➽⣽⬊ࢡࢳࣥࡢྠᐃFLJ00018
ࡢ᪂つ⤖ྜ⺮ⓑ㉁ࢆྠᐃࡍࡿⅭࠊFLJ00018
ࡢGEF
άᛶ㔜せ࡞DH
ཬࡧPH
ࢻ࣓ࣥࢆྵࡴ
1-465
␒┠ࡢ࣑ࣀ㓟㓄ิࢆ⏝࠸ࠊ㓝ẕtwo-hybrid
ἲࢆ⾜ࡗࡓࠋbaitࡋ࡚⏝࠸ࡓ
FLJ00018 1-465
࣑ࣀ㓟ࢆᑟධࡋࡓ㓝ẕY187
ᰴࠊࣄࢺ⫾ඣ⬻cDNA
ࡽᚓࡽࢀࡓFLJ00018
⤖ྜೃ⿵⺮ⓑ㉁ࡢⓎ⌧࣋ࢡࢱ࣮ࡢࣛࣈ࣮ࣛࣜࢆᑟධࡋࡓ㓝ẕY2H Gold
ᰴࢆ᥋ྜࡉࡏࡓࠋ᥋ྜࡋࡓ
9.010
6ࢡ࣮ࣟࣥࡢ㓝ẕࢆࢺࣜࣉࢺࣇࣥཬࡧࣟࢩࣥࢆḞࡃSD
ᇵᆅୖ࡛ᇵ㣴ࡋࠊࢫࢡ࣮ࣜࢽࣥࢢࢆ࠾ࡇ࡞ࡗࡓࡇࢁࠊ1424ಶࡢ㝧ᛶࢡ࣮ࣟࣥࡀᚓࡽࢀࡓࠋࡇࢀࡽࡍ
࡚ࢆࡉࡽ㑅ᢥᛶࡢ㧗࠸ࠊࢹࢽࣥࠊࣄࢫࢳࢪࣥࠊࢺࣜࣉࢺࣇࣥࠊࣟࢩࣥࢆḞࡃ
SD
ᇵᆅୖ࡛ᇵ㣴ࢆ⾜ࡗࡓࡇࢁࠊ971 ࡢ㝧ᛶࢡ࣮ࣟࣥࢆᚓࡓࠋᚓࡽࢀࡓࢡ࣮ࣟࣥࡽ
285
ಶࢆ↓సⅭ㑅ᢥࡋ㑇ఏᏊᢳฟࢆ⾜࠸ࠊ
FLJ00018 1-465
ࡢ㓄ิඹࡧY2H Gold
ᰴࢺࣛࣥࢫࣇ࢛࣮࣒ࡍࡿࡇ࡛ࠊೃ⿵㑇ఏᏊࡢ⮬❧άᛶ⬟ࡢ᭷↓ࡢ☜ㄆࢆ⾜ࡗࡓࠋࡇࢀࡽࡢࢫࢡ࣮ࣜࢽࣥࢢࡼࡗ
࡚ᚓࡽࢀࡓ┿ࡢ㝧ᛶࢡ࣮ࣟࣥ
121
ಶࡢ㓄ิ☜ㄆࢆ⾜ࡗࡓࠋFigure 3-1
ࠊࢡ࣮ࣟࣥ␒ྕ11-2
ࡢ⮬❧άᛶ⬟ࢆヨ㦂ࡋࡓ⤖ᯝࢆ♧ࡍࠋᕥୗࡢ┿♧ࡍࡼ࠺ࠊࢥࣥࢺ࣮ࣟࣝ࣋ࢡࢱ࣮
11-2
ࢆᙧ㉁㌿ࡋࡓ㓝ẕࡣ㑅ᢥᇵᆅ⏕Ꮡ࡛ࡁࡎࠊྑୗࡢ┿♧ࡍࡼ࠺ࠊbait
11-2
ࡢᙧ㉁㌿ࡣᏑᅾ࡛ࡁࡿࡇࡽࠊ㝧ᛶ࡛࠶ࡿࡇࡀ☜ㄆ࡛ࡁࡿࠋ Fig. 3--1 FLLJ00018 1-4465aa (Bait) ࢡ࣮ࣟࣥ11-2 (Prey) ࡢ┦స⏝
pGBKT7-FLJ00018 1-465aa ࡲ ࡓ ࡣ ࠊ pGBKT7-DNA-BDࢆpGADT7-11-2ࡶ
㓝ẕ Y2H Gold ᰴࢺࣛࣥࢫࣇ࢛࣮࣒ࡋᖹᯈ
ᇵᆅ࡛ᇵ㣴ࡋࡓࠋୖẁ(DDO/X);ࢺࣜࣉࢺࣇ
ࣥ ࠊ ࣟ ࢩ ࣥ ࢆ 㝖 ࠸ ࡓ SD ᇵ ᆅ ࠊ ୗ ẁ
(QDO/X/A); ࢺࣜࣉࢺࣇࣥࠊࣟࢩࣥࠊࢹ
ࢽࣥࠊࣄࢫࢳࢪࣥࢆ㝖࠸ࡓSDᇵᆅࠋ
54
ࡇࡢ㑇ఏᏊࡢ㓄ิࢆ
GenBank
ࡢࢹ࣮ࢱ࣮࣋ࢫୖࡢ㓄ิẚ㍑ࡋࡓࡇࢁࠊ㠀➽⣽⬊࠾࠸࡚Ⓨ⌧ࡍࡿJ-ࢡࢳࣥ㧗࠸┦ྠᛶࢆ᭷ࡍࡿࡇࡀ᫂ࡽ࡞ࡗࡓࠋ
22.
ᇵ㣴⣽⬊ෆ࠾ࡅࡿFLJ00018
J-ࢡࢳࣥཬࡧE-ࢡࢳࣥࡢ┦స⏝⬨᳝ື≀࠾ࡅࡿࢡࢳࣥࡣࠊ6ࡘࡢࢯࣇ࢛࣮࣒ࡀᏑᅾࡋࠊᶓ⣠➽ࠊ㦵᱁➽ཬࡧࠊ㠀➽⣽
⬊ࡢ⣽⬊㉁࡛Ⓨ⌧ࡍࡿࢯࣇ࢛࣮࣒ࡀࡑࢀࡒࢀ
2
ࡘࡎࡘᏑᅾࡍࡿࡇࡀ▱ࡽࢀ࡚࠸ࡿࠋࡑࢀࡒࢀࡢࢯࣇ࢛࣮࣒ࡣ㧗࠸┦ྠᛶࢆ♧ࡋࠊ≉ࠊ㠀➽⣽⬊ࡢ⣽⬊㉁࡛Ⓨ⌧ࡍࡿࢯࣇ࢛࣮࣒
࡛࠶ࡿJ-ࢡࢳࣥE-ࢡࢳࣥࡣ
N
ᮎ➃ࡢ1, 2, 3
ࡑࡋ࡚9
␒┠ࡢ࣑ࣀ㓟ࢆ㝖ࡃ㓄ิࡣ➼ࡋ࠸(84)ࠋᅇ two-hybrid
࡛ᚓࡽࢀࡓ㓄ิࡣJ-ࢡࢳࣥࡢ257-376
␒┠ࡢ࣑ࣀ㓟㧗࠸┦ྠᛶࢆ♧ࡋࠊJ-ࢡࢳࣥࡢ
257-376
␒┠ࡢ㓄ิࡣࠊE-ࢡࢳࣥࡶ┦ྠᛶࢆ᭷ࡍࡿ (Fig. 3-2A)ࠋࡑࡇ࡛ࠊ
Flag-ࢱࢢࢆຍࡋࡓJ-ࢡࢳࣥཬࡧࠊ
E-ࢡࢳࣥࢆసᡂࡋࠊMyc
ࢱࢢࢆຍࡋࡓFLJ00018
ඹ
HEK293
⣽⬊ඹⓎ⌧ࡉࡏࠊචỿ㝆ࢆ⾜࠺ࡇࡼࡾࠊᇵ㣴⣽⬊ෆ࠾ࡅࡿ┦స⏝ࢆ☜ㄆࡋࡓࠋࡑࡢ⤖ᯝࠊ
Figure 3-2B
♧ࡍᵝࠊFLJ00018 WT
ඹỿ㝆ࡍࡿJ-ࢡࢳࣥཬࡧࠊ E-ࢡࢳࣥࡀぢࡽࢀࠊFLJ00018ࡣ့ங㢮⣽⬊࠾࠸࡚ࡶJ-ࢡࢳࣥཬࡧࠊE-ࢡࢳࣥ┦స⏝ࡍࡿࡇࡀ♧၀ࡉࢀࡓࠋࡲࡓࠊ㓝ẕ
two-hyabrid
ἲ࡛⏝࠸ࡓFLJ00018
ࡢ1-465
␒┠ࡢ࣑ࣀ㓟㓄ิࡘ࠸࡚ࡶྠᵝJ-ࢡࢳࣥཬࡧࠊE-ࢡࢳࣥ┦స⏝ࡍࡿࡇࡀ᫂ࡽ࡞ࡗࡓ
(Fig. 3-2C)ࠋ
ࡉࡽࠊෆᅉᛶࡢE-ࢡࢳࣥࡢ┦స⏝ࢆ᳨ウࡍࡿࡓࡵࠊ
FLJ00018
ࢆ༢⊂࡛⣽⬊Ⓨ⌧ࡉࡏ චỿ㝆≀ࢆゎᯒࡋࡓ⤖ᯝࠊFLJ00018 WTཬࡧFLJ00018 P2
ࡢඹỿ㝆≀ෆE-ࢡࢳࣥࡢ ࢩࢢࢼࣝࡀ☜ㄆࡉࢀࠊ⏕⌮ⓗ᮲௳ୗ࡛ࡶFLJ00018
ࡀE-ࢡࢳࣥ┦స⏝ࡍࡿࡇࡀ♧၀ࡉࢀࡓ (Fig. 3-2D)ࠋ
55
33. E-ࢡࢳࣥࡢ┦స⏝㛵ࢃࡿ FLJ00018
ୖࡢ㓄ิࡢྠᐃFLJ00018
ࡀࠊtwo-hybrid
࡛⏝࠸ࡓࠊ1-465
࣑ࣀ㓟ࡢ㡿ᇦ௨እ࠾࠸࡚┦స⏝ࡍࡿࢆ᳨ウࡍࡿⅭࠊFLJ00018ࡢ
449-1386
␒┠ࡢ࣑ࣀ㓟㓄ิࢆࢥ࣮ࢻࡍࡿFLJ00018
'NTኚ␗యࢆసᡂࡋ (Fig 3-3A)ࠊචỿ㝆ἲࢆ⏝࠸࡚E-ࢡࢳࣥࡢ┦స⏝ࢆゎᯒࡋࡓࠋ
Figure 3-3B
♧ࡍᵝࠊFLJ00018 'NT ࡣࠊE-ࢡࢳࣥඹỿ㝆ࡋࠊ1-465 ␒┠ࡢ࣑ࣀ㓟㓄ิ௨እࡶ
Fiig. 3--2 FLJ00018㠀➽⣽⬊ࢡࢳࣥࡢ┦స⏝
A, FLJ00018ཬࡧ㠀➽⣽⬊ࢡࢳࣥࡢᵓ㐀ࠋDH, dbl ࣍ࣔࣟࢪ࣮ࢻ࣓ࣥ; PH, pleckstrin ࣍ࣔࣟࢪ
࣮ࢻ࣓ࣥ; Y2H, yeast two-hybrid preyࢥࣥࢫࢺࣛࢡࢺ
B-C, HEK293⣽⬊MycࢱࢢࢆຍࡋࡓFLJ00018 WT (B) ཪࡣࠊFLJ00018 P2 (C) ࢆࠊFlag-ࢱࢢ
ࢆຍࡋࡓE-ࢡࢳࣥཬࡧJ-ࢡࢳࣥඹᅗ♧ࡋࡓࡼ࠺୍㐣ᛶࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡋࡓࠋ⣽
⬊ࡢ◚○ᚋࠊᢠMycᢠయࢆ⏝࠸࡚FLJ00018ࢆචỿ㝆ࡋࡓࠋචỿ㝆ᚋࠊSDS-PAGE࡛ศ㞳ࡋࡓࠋ
࣒ࣀࣈࣟࢵࢺࡣࠊMycࢱࢢࡀຍࡉࢀࡓFLJ00018ࡢ᳨ฟࡢⅭࠊAnti-Mycᢠయࢆ⏝࠸ࠊFlagࢱ ࢢࡀຍࡉࢀࡓࢡࢳࣥࡢ᳨ฟࡢⅭࠊAnti-Flagᢠయࢆ⏝࠸ࡓࠋ
D, HEK293⣽⬊MycࢱࢢࢆຍࡋࡓFLJ00018WTཪࡣࠊFLJ00018 P2ࢆᅗ♧ࡋࡓࡼ࠺୍㐣 ᛶࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡋࡓࠋ⣽⬊ࡢ◚○ᚋࠊᢠMycᢠయࢆ⏝࠸࡚FLJ00018ࢆචỿ㝆ࡋࡓࠋච
ỿ㝆ᚋࠊSDS-PAGE࡛ศ㞳ࡋࡓࠋ࣒ࣀࣈࣟࢵࢺࡣࠊMycࢱࢢࡀຍࡉࢀࡓFLJ00018ࡢ᳨ฟࡢⅭ
ࠊAnti-Myc ᢠయࢆ⏝࠸ࠊࢡࢳࣥࡢ᳨ฟࡢⅭࠊAnti-E-ࢡࢳࣥᢠయࢆ⏝࠸ࡓࠋ(*)ࡣࠊචỿ㝆
ຍ࠼ࡓMyc-ᢠయ⏤᮶ࡍࡿ㠀≉␗࡞⤖ྜࢆ♧ࡍࠋ
56
FLJ00018
ࢡࢳࣥࡢ┦స⏝ࡍࡿ㓄ิࡀ࠶ࡿࡇࡀ♧၀ࡉࢀࡓࠋࡇࢀࡽࡢ⤖ᯝࡽࠊFLJ00018
ࢡࢳࣥࡢ┦స⏝㓄ิࡣࠊᑡ࡞ࡃࡶ2
⟠ᡤᏑᅾࡍࡿࡇࡀ♧၀ࡉࢀࡓࠋFLJ00018
ࡢ1-465
␒┠ࡢ㡿ᇦ࠾ࡅࡿ┦స⏝㒊ࡢヲ⣽ࡘ࠸࡚ࡉࡽゎᯒࢆ㐍ࡵࡿⅭࠊ1-465aa
ࡢN
ᮎ➃ཬࡧC
ᮎ➃ࡽ㡰Ḟᦆࡉࡏࡓኚ␗యࢆసᡂࡋࡓ (Fig. 3-4A)ࠋస〇ࡋࡓFLJ00018
ࡢྛ✀ኚ␗యࡢ࠺ࡕࠊࡲࡎP2 (Bait) ྛ✀ N
ᮎ➃Ḟᦆኚ␗య࡛࠶ࡿP2
'N1ኚ␗యࠊ
P2 'N2
ኚ␗యࠊP2 'N3
ኚ␗యE-ࢡࢳࣥࡢ┦స⏝ࡘ࠸᳨࡚ウࢆ⾜ࡗࡓࠋࡑࡢ⤖ᯝࠊBait
ྠᵝࡢ㓄ิࢆࡶࡘF018 P2
ࡢࠊP2 'N1ཬࡧP2
'N2࡛ࡣE-ࢡࢳࣥࡢ┦స⏝ࡀぢࡽࢀࡓࡀࠊ
P2 'N3
࡛ࡣE-ࢡࢳࣥࡢ┦స⏝ࡣぢࡽࢀ࡞ࡗࡓ (Fig. 3-4B)ࠋḟࠊbait
ࡋ࡚⏝ࡋࡓ
P2
ኚ␗యෆࡢC
ᮎ➃ഃࢆḞᦆࡉࡏࡓP2 'C1, P2 'C2, P2 'C3
ཬࡧPH
ኚ␗య FFiig. 33--33 FFLJ00018 ''NNTኚኚ␗య㠀➽⣽⬊ࢡࢳࣥࡢ┦స⏝
A, FLJ00018 'NTኚ␗యࡢᵓ㐀ࠋFLJ00018 'NTࡣFLJ00018ࡢ449-1386␒┠ࡲ࡛ࡢ࣑ࣀ㓟ࢆࢥ
࣮ࢻࡍࡿࠋ
B, HEK293⣽⬊MycࢱࢢࢆຍࡋࡓFLJ00018 WT ࡲࡓࡣࠊFLJ00018 P2, FLJ00018 'NTࢆࠊ
Flag-ࢱࢢࢆຍࡋࡓE-ࢡࢳ୍ࣥ㐣ᛶࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡋࡓࠋ⣽⬊ࡢ◚○ᚋࠊᢠMycᢠయࢆ
⏝࠸࡚ FLJ00018 ࢆචỿ㝆ࡋࡓࠋචỿ㝆ᚋࠊSDS-PAGE ࡛ศ㞳ࡋࡓࠋ࣒ࣀࣈࣟࢵࢺࡣࠊMyc
ࢱࢢࡀຍࡉࢀࡓFLJ00018ࡢ᳨ฟࡢⅭࠊAnti-Mycᢠయࢆ⏝࠸ࠊFlagࢱࢢࡀຍࡉࢀࡓࢡࢳࣥ
ࡢ᳨ฟࡢⅭࠊAnti-Flagᢠయࢆ⏝࠸ࡓࠋ(*)ࡣࠊචỿ㝆ຍ࠼ࡓMyc-ᢠయ⏤᮶ࡍࡿ㠀≉␗࡞⤖
ྜࢆ♧ࡍࠋ
57
E-ࢡࢳࣥࡢ┦స⏝ࡘ࠸᳨࡚ウࢆ⾜ࡗࡓࠋࡑࡢ⤖ᯝࠊP2 'C1, P2 'C2ཬࡧࠊP2 'C3
࡛ࡣE-ࢡࢳࣥࡢ┦స⏝ࡀ☜ㄆ࡛ࡁࡓࡀࠊ
PH
࡛ࡣ┦స⏝ࡀ☜ㄆ࡛ࡁ࡞ࡗࡓ (Fig. 3-4C)ࠋ ௨ୖࡢ⤖ᯝࡼࡾࠊFLJ00018 P2
ෆ࡛ࡣFLJ00018
ࡢάᛶ㔜せ࡞DH
ࢻ࣓ࣥࢆྵࡴ150
␒┠ࡽ
283
␒┠ࡢ࣑ࣀ㓟㓄ิࡀࠊE-Actinࡢ┦స⏝ᚲせ࡞㓄ิ࡛࠶ࡿࡇࡀ♧၀ࡉࢀࡓࠋ44. E-ࢡࢳࣥ࠾ࡼࡧJ-ࢡࢳࣥࡼࡿ FLJ00018
ࢆࡋࡓSRE
౫Ꮡⓗ㌿άᛶࡢᢚไFLJ00018
ࢡࢳࣥࡢ┦స⏝ࡀFLJ00018
ࡢάᛶࡢࡼ࠺࡞ᙳ㡪ࢆ࠼ࡿࡢࡘ࠸࡚ㄪࡿࡓࡵࠊE-ࢡࢳࣥ࠾ࡼࡧࠊJ-ࢡࢳࣥࢆࠊ
FLJ00018 WT
࠾ࡼࡧࠊP2
ࡑࢀࡒࢀඹFiig. 3--4 FLJ00018P2㡿㡿ᇦ࠾ࡅࡿ㠀➽⣽⬊ࢡࢳࣥࡢ┦స⏝㡿ᇦࡢྠᐃ
A, FLJ00018 P2ࡢྛ✀ኚ␗యࡢᵓ㐀ࠋP2, P2'N1, P2'N2, P2'N3, P2'C1, P2'C2, P2'C3, PHࡣࡑ
ࢀࡒࢀFLJ00018ࡢ1-464, 58-464, 150-464, 283-464, 1-441, 1-355, 1-310ࡑࡋ࡚283-441␒┠ࡲ࡛ࡢ
࣑ࣀ㓟ࢆࢥ࣮ࢻࡋ࡚࠸ࡿࠋ
B, C, MycࢱࢢࢆຍࡋࡓFLJ00018ࡢྛ✀ኚ␗యFlag-ࢱࢢࢆຍࡋࡓE-ࢡࢳࣥࢆHEK293⣽
⬊ෆ୍㐣ᛶࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡋࡓࠋ⣽⬊ࡢ◚○ᚋࠊᢠMycᢠయࢆ⏝࠸࡚FLJ00018ࢆචỿ 㝆ࡋࡓࠋචỿ㝆ᚋࠊSDS-PAGE࡛ศ㞳ࡋࡓࠋ࣒ࣀࣈࣟࢵࢺࡣࠊMycࢱࢢࡀຍࡉࢀࡓFLJ00018 ࡢ᳨ฟࡢⅭࠊAnti-Mycᢠయࢆ⏝࠸ࠊFlagࢱࢢࡀຍࡉࢀࡓࢡࢳࣥࡢ᳨ฟࡢⅭࠊAnti-Flagᢠయ
ࢆ⏝࠸ࡓࠋ(*)ࡣࠊචỿ㝆ຍ࠼ࡓMyc-ᢠయ⏤᮶ࡍࡿ㠀≉␗࡞⤖ྜࢆ♧ࡍࠋ
58
Ⓨ⌧ࡉࡏ
FLJ00018
άᛶࡢᣦᶆ࡛࠶ࡿSRE
άᛶ ᐃࢆ⾜࡞ࡗࡓࠋࡑࡢ⤖ᯝࠊࡑࢀࡒࢀࡢ⺮ⓑ㉁ࡢⓎ⌧ᕪ␗ࡀぢࡽࢀ࡞࠸ࡶࢃࡽࡎࠊE-ࢡࢳࣥ
FLJ00018
ࡢඹⓎ⌧ࡼࡾࠊFLJ00018ࢆࡋࡓSRE
άᛶࡀࡁࡃᢚไࡉࢀࡓࠋE-ࢡࢳࣥJ-ࢡࢳࣥࢆẚ㍑ࡍࡿࠊE-ࢡࢳࣥࡼࡿ
FLJ00018
ࢆࡋࡓSRE
άᛶࡢᢚไຠᯝࡣࠊJ-ࢡࢳࣥࡼࡿᢚไຠᯝࡼࡾࡁࡗࡓࠋ୍᪉ࠊFLJ00018 ࡣࠊ୕㔞య
G
⺮ⓑ㉁ࡢGEJࢧࣈࣘ
ࢽࢵࢺࡼࡗ࡚άᛶࡉࢀࡿࡇࡽࠊGEJࢧࣈࣘࢽࢵࢺࡼࡿ
FLJ00018
ࡢSRE
άᛶୖ᪼ᑐࡍࡿࢡࢳࣥࡢᙳ㡪ࢆ᳨ウࡋࡓࠋࡑࡢ⤖ᯝࠊࢡࢳࣥ
FLJ00018
࠾ࡼࡧGEJࢧࣈࣘࢽࢵࢺ
ࢆඹⓎ⌧ࡉࡏࡓ⣽⬊࡛ࡣࠊFLJ00018 ࢡࢳࣥࡔࡅࢆඹⓎ⌧ࡉࡏࡓ⣽⬊ẚࠊ᭷ព
SRE
άᛶୖ᪼ࡢᢚไࡀぢࡽࢀࡓ (Fig. 3-5)ࠋ59
E-ࢡࢳࣥࡀ
FLJ00018
ࡢάᛶࢆᢚไࡍࡿࡇࢆ☜ࡵࡿⅭࠊ⭠⳦DH5Dࡼࡾ⢭〇ࡋࡓ GST-Rac G15A
ࢆ⏝࠸ࡓpull-down
ࢵࢭࡼࡾࠊFLJ00018ࡢRhoGTPase
ᑐࡍࡿGEF
άᛶࢆホ౯ࡋࡓࠋRac G15A
ࡣࠊRac
ࡢࢾࢡࣞ࢜ࢳࢻࣇ࣮ࣜኚ␗య࡛࠶ࡾࠊࡇࡢኚ␗య⤖ྜࡍࡿ
RhoGEF
ࡢ㔞ࡣࠊάᛶࡋࡓRhoGEF
ࡢᣦᶆࡍࡿࡇࡀ࡛ࡁࡿࡉࢀࡿ (83)ࠋFigure 3-6
♧ࡋࡓࡼ࠺ࠊ
FLJ00018
ࡢࡳࢆⓎ⌧ࡉࡏ⣽⬊ẚࠊFLJ00018E-ࢡࢳࣥࢆඹⓎ⌧ࡉࡏ ࡓ⣽⬊࠾࠸࡚ࠊGST-Rac G15Aඹỿ㝆ࡍࡿFLJ00018
ࡢ㔞ࡢῶᑡࡀࡳࡽࢀࡓࠋ௨ୖࡢ⤖ᯝࡼࡾࠊFLJ00018ࢡࢳࣥࡢ┦స⏝ࡀ
FLJ00018
ࡢάᛶࢆᢚไࡍࡿྍ⬟ᛶࡀ♧၀ࡉࢀࡓࠋ FFiig. 33--55 ࢡࢳࣥࡀFFLJ00018ࢆࢆࡋࡓSSRE౫౫Ꮡⓗ㌿άᛶ࠼ࡿᙳ㡪
A, C, HEK293 ⣽ ⬊ pSRE.L-luciferase, pRL-SV40 ࣉ ࣛ ࢫ ࣑ ࢻ DNA, FLJ00018 WT (A), FLJ00018 P2 (C), GE, GJ, E-ཬࡧJ-ࢡࢳࣥࡢⓎ⌧࣋ࢡࢱ࣮ࢆࡑࢀࡒࢀᅗ♧ࡋࡓࡼ࠺ࢺࣛࣥࢫࣇ
࢙ࢡࢩࣙࣥࡋࡓࠋࣝࢩࣇ࢙࣮ࣛࢮάᛶࡣdual-luciferase reporter assay systemࡼࡗ࡚ ᐃࡋࠊᚓ
ࡽࢀࡓάᛶࡣMockࢆ1.0ࡋ࡚ᶆ‽ࡋࡓ್ࢆ♧ࡋࡓࠋᐇ㦂ࡣᑡ࡞ࡃࡶ3ᅇ⾜࠸ࠊ್ࢆᖹᆒ್±
S.D.࡛♧ࡋࡓࠋ
B, D, HEK293⣽⬊, MycࢱࢢࢆຍࡋࡓFLJ00018 WT (B)ࡲࡓࡣFLJ00018 P2 (D)ࠊFlagࢱࢢ
ࢆຍࡋࡓE-ཬࡧJ-ࢡࢳࣥࠊࡑࡋ࡚GE, GJࡢⓎ⌧࣋ࢡࢱ࣮ࢆᅗ♧ࡋࡓࡼ࠺୍㐣ᛶࢺࣛࣥࢫࣇ
࢙ࢡࢩࣙࣥࡋࡓࠋࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥᚋࠊ➼㔞ࡢ⺮ⓑ㉁ࢆ7.5% SDS-PAGE࡛ศ㞳ࡋࡓࠋ Myc ࢱ ࢢ ࡀ ຍ ࡉ ࢀ ࡓ FLJ00018 ࡢ ᳨ ฟ ࡢ Ⅽ ࠊAnti-Myc ᢠ య ࢆ ⏝ ࠸ ࠊFLJ00018 ࡢ ᳨ ฟ ࡣ ࠊ Anti-PLEKHG2 ᢠయࢆ⏝࠸ࡓࠋFlag ࢱࢢࡀຍࡉࢀࡓE-ཬࡧJ-ࢡࢳࣥࡢ᳨ฟࡣࠊAnti-Flag ᢠ యࢆ⏝࠸ࠊE-ࢡࢳࣥࡢ᳨ฟࡣࠊAnti-E-ࢡࢳࣥᢠయࢆ⏝࠸ࡓࠋࡉࡽࠊGEࢧࣈࣘࢽࢵࢺࡢ᳨ฟ
ࠊAnti-GEᢠయࢆ⏝ࡋࡓࠋ
F
Fiig. 33--66 ࢡࢳࣥࡀFFLJ00018ࡢࡢάᛶ࠼ࡿᙳ㡪 HEK293⣽⬊, MycࢱࢢࢆຍࡋࡓFLJ00018 WTࠊ Flag ࢱࢢࢆຍࡋࡓE-ࢡࢳࣥࢆᅗ♧ࡋࡓࡼ࠺
୍㐣ᛶࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡋࡓࠋࢺࣛࣥࢫࣇ࢙ࢡ ࢩࣙࣥᚋࠊ⣽⬊⁐ゎᾮࠊࢭࣇ࣮ࣟࢫࣅ࣮ࢬࡀ⤖ྜ
ࡋࡓGST-Rac G15Aࢆຍ࠼ࠊᛂࢆ⾜ࡗࡓࠋࣅ࣮ࢬ
⤖ྜࡋࡓ⺮ⓑ㉁ࢆ SDS-PAGE ࡛ศ㞳ࡋࠊࡑࢀࡒࢀࠊ ᅗ♧ࡋࡓᢠయࢆ⏝࠸࡚࣒ࣀࣈࣟࢵࢺࢆ⾜ࡗࡓࠋ
60
55. E-ࢡࢳࣥࡀ FLJ00018
GEJࡢ┦స⏝࠼ࡿᙳ㡪
㠀➽⣽⬊ࢡࢳࣥࡀ
FLJ00018
ࢆࡋࡓSRE
౫Ꮡⓗ㌿άᛶࢆᢚไࡍࡿࡇࡽࠊFLJ00018
GEJࡢ┦స⏝ࢡࢳࣥࡀ࠼ࡿᙳ㡪ࡘ࠸᳨࡚ウࢆ⾜ࡗࡓࠋFLJ00018
GEJཬࡧE-ࢡࢳࣥࢆ
HEK293
⣽⬊ࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡋࠊචỿ㝆ࡼࡾࠊFLJ00018┦స⏝ࡍࡿ
GEJཬࡧE-ࢡࢳࣥࢆ᳨ウࡋࡓࠋFigure 3-7
♧ࡍᵝFLJ00018
ඹỿ㝆ࡍࡿGEJࡢ㔞
ࡣࠊE-ࢡࢳࣥࡢඹⓎ⌧ࡼࡾࠊኚࡣぢࡽࢀ࡞ࡗࡓࠋࡇࡢࡇࡽE-ࢡࢳࣥGEJࡢ
FLJ00018
ࡢ┦స⏝㛵ࢃࡿ㓄ิࡣ␗࡞ࡿྍ⬟ᛶࡀ♧၀ࡉࢀࡓࠋ6. FLJ00018
ࢡࢳࣥࡢ⣽⬊ෆ࠾ࡅࡿᒁᅾ⣽⬊ෆ࠾ࡅࡿ
FLJ00018
ෆᅉᛶࡢࢡࢳࣥࡢᒁᅾࡘ࠸᳨࡚ウࡍࡿⅭࠊNIH3T3 ⣽⬊FLJ00018
ཬࡧGEJࢆ୍㐣ᛶⓎ⌧ࡉࡏࡓ (Fig. 3-8A, B)ࠋࡇࡢ⣽⬊ࢆ⏝࠸ࠊ⣽⬊චᰁⰍࢆ⾜
࠸ࠊ
FLJ00018
ࢡࢳࣥࡢࡑࢀࡒࢀࡢᒁᅾࡘ࠸᳨࡚ウࡋࡓࡇࢁࠊFLJ00018
GEJࢆඹⓎ
⌧ࡉࡏࡓ⣽⬊࠾࠸࡚ࠊFLJ00018 ࡣ⥺⥔≧ࡢ
F-ࢡࢳࣥඹᒁᅾࡀࡳࡽࢀࡓ(Fig. 3-8C)ࠋ୍
᪉ࠊFLJ00018 ࡢࡳࢆⓎ⌧ࡉࡏࡓ⣽⬊࡛ࡣࠊFLJ00018 ࡣ⣽⬊⭷㏆ࡢ⣽⬊ࡢ⦕࠾࠸࡚
F-
ࢡࢳࣥᙅࡃ⤖ྜࡋ࡚࠸ࡓࠋࡇࢀࡽࡢ⤖ᯝࡽࠊFLJ00018 ࡣࠊ⥺⥔≧
F-ࢡࢳࣥ┦స⏝
ࡍࡿࡇࡀ♧၀ࡉࢀࡓࠋ
Fiig. 3--7 FLJ00018GGEJJࡢ⤖ྜEE-ࢡࢳࣥࡀ
࠼ࡿᙳ㡪
MycࢱࢢࢆຍࡋࡓFLJ00018Flag-ࢱࢢࢆ
ຍࡋࡓE-ࢡࢳࣥࠊGEཬࡧGJࢆHEK293⣽⬊ෆ
୍㐣ᛶࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡋࡓࠋ⣽⬊ࡢ◚
○ᚋࠊᢠMycᢠయࢆ⏝࠸࡚FLJ00018ࢆචỿ㝆 ࡋࡓࠋචỿ㝆ᚋࠊSDS-PAGE࡛ศ㞳ࡋࠊᅗ♧
ࡋࡓᢠయࢆ⏝࠸࡚࣒ࣀࣈࣟࢵࢺࢆ⾜ࡗࡓࠋ
61
62
Fiig. 3--8 FLJ00018ࡣࡣෆᅉᛶࡢ⥺⥔≧ࢡࢳࣥඹᒁᅾࡍࡿ
A, NIH3T3⣽⬊ࠊMycࢱࢢࢆຍࡋࡓFLJ00018 WT ཬࡧGE, GJࡢⓎ⌧࣋ࢡࢱ࣮ࢆᅗ♧ࡋࡓࡼ
࠺୍㐣ᛶࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡋࡓࠋࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥᚋࠊ➼㔞ࡢ⺮ⓑ㉁ࢆSDS-PAGE࡛ ศ㞳ࡋࡓࠋ Myc ࢱࢢࡀຍࡉࢀࡓFLJ00018ࡢ᳨ฟࡢⅭࠊAnti-Mycᢠయࢆ⏝࠸ࠊGEࢧࣈࣘࢽࢵ
ࢺࡢ᳨ฟࠊAnti-GEᢠయࢆ⏝ࡋࡓࠋ
B, C, NIH3T3⣽⬊ࠊMycࢱࢢࢆຍࡋࡓFLJ00018 WT ཬࡧGE, GJࡢⓎ⌧࣋ࢡࢱ࣮ࢆᅗ♧ࡋ ࡓࡼ࠺୍㐣ᛶࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡋࡓࠋࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥᚋࠊ⣽⬊ࢆᅛᐃࡋࠊGE (B)ࡲࡓ ࡣࠊFLJ00018 (C)F-ࢡࢳࣥࢆ㔜ᰁⰍࡋࡓࠋࢫࢣ࣮ࣝࣂ࣮, 20 Pm
63
➨
➨ 4 ⠇ ⪃ᐹ
ᮏ◊✲࠾࠸࡚ࠊGEJ౫Ꮡⓗ
RhoGEF
࡛࠶ࡿFLJ00018
ࡀࠊGEJ௨እࠊࢡࢳࣥ⤖ྜࡍࡿࡇࢆぢฟࡋࡓࠋFLJ00018ࢡࢳࣥࡢ┦స⏝㒊ࡣ
2
⟠ᡤ௨ୖ࠶ࡿࡇࡀ♧၀ࡉࢀࠊtwo-hybrid
ࢫࢡ࣮ࣜࢽࣥࢢཬࡧࠊචỿ㝆ࡢ⤖ᯝࡼࡾࠊFLJ00018ࡢࢡࢳࣥ⤖ྜ㒊ࡢ1
ࡘ ࡣࠊ150-283
␒┠ࡢ࣑ࣀ㓟Ꮡᅾࡍࡿࡇࡀ᫂ࡽ࡞ࡗࡓࠋ㐣ཤࠊFLJ00018ࡣࠊN
ᮎ➃㏆ࡢ㓄ิ࠾࠸࡚ࠊ
GEJ┦స⏝ࡍࡿࡇࡀ♧ࡉࢀ࡚࠸ࡿࠋࡋࡋࠊࢡࢳࣥ FLJ00018
ࡢ⤖ྜࡣࠊ
FLJ00018
GEJࡢ┦స⏝ᙳ㡪ࢆ࠼࡞࠸ࡇࡀ♧၀ࡉࢀࡓࠋ᭦ࠊᮏ◊✲࠾
࠸࡚ࠊFLJ00018ࡣࠊ⥺⥔≧ࡢE-ࢡࢳࣥ⤖ྜࡋࠊࢡࢳࣥࡢ⤖ྜࡣࠊFLJ00018ࡢάᛶ
ᑐࡋࠊ㈇ࡢไᚚࢆࡍࡿࡇࡀ᫂ࡽ࡞ࡗࡓ (Fig. 3-9)ࠋ
ࢡࢳࣥࡢ⤖ྜࡼࡿ
FLJ00018
ࡢάᛶᢚไࡣࠊࢡࢳࣥࡢࢯࣇ࢛࣮࣒ࡼࡗ࡚㐪࠸ࡀぢࡽࢀࠊJ-ࢡࢳࣥࡼࡾࡶE-ࢡࢳࣥࡢ⤖ྜࡼࡿ
FLJ00018
ࡢάᛶᢚไຠᯝࡢ᪉ࡀᙉ࠸ࡇࡀᮏ◊✲࡛♧၀ࡉࢀࡓࠋ࠸ࡃࡘࡢㄽᩥ࠾࠸࡚ࠊE-ࢡࢳࣥJ-ࢡࢳࣥࡢ⣽⬊ෆᒁᅾࡀ␗
࡞ࡿ࠸࠺ሗ࿌ࡀ࡞ࡉࢀ࡚࠸ࡿ (84,85)ࠋࡇࡢࡇࡽࠊFLJ00018 E-ࢡࢳࣥ࠾ࡼࡧJ-ࢡ ࢳࣥࡢ⣽⬊ᒁᅾࡢ㐪࠸ࡀࠊE-ࢡࢳࣥ࠾ࡼࡧJ-ࢡࢳࣥࡢ
FLJ00018
ࡢάᛶᢚไᑐࡍࡿຠᯝࡢ 㐪࠸⾲ࢀ࡚࠸ࡿࡇࡶ⪃࠼ࡽࢀࡿࠋ୍᪉ࠊᮏ◊✲࡛ࡣࠊin vitro
࠾ࡅࡿFLJ00018
ࢡࢳࣥࡢ┤᥋ⓗ࡞┦స⏝ࢆ☜ㄆࡍࡿࡇࡀ࡛ࡁ࡞ࡗࡓࠋࡲࡓࠊE-ࢡࢳࣥ࠾ࡼࡧJ-ࢡࢳࣥࡑࢀ
ࡒࢀࡢࢯࣇ࢛࣮࣒ࢆ⏝࠸ࡓ
FLJ00018
ࡢᒁᅾࡢ㐪࠸ࢆ☜ㄆࡍࡿࡇࡶ࡛ࡁ࡞ࡗࡓࠋᚋࡉࡽ࡞ࡿ᳨ウࡀᚲせ⪃࠼ࡽࢀࡿࠋ
᭱㏆ࠊ㠀➽⣽⬊ࡢ
myosinϩࡀE-Pix
ࢆྵࡴ」ᩘࡢDbl
ࣇ࣑࣮ࣜࡢRhoGEF
⤖ྜࡍࡿࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿࠋmyosinϩࡢ⤖ྜࡣ
DH, PH
ࢻ࣓ࣥ㏆ࡢᵓ㐀ࡀ㛵ࡋࠊ⤖ྜࡼࡗ࡚
RhoGEF
ࡢάᛶࡀᢚไࡉࢀࡿ(86)ࠋࡲࡓࠊE-Pix-bLࡣࢳࣗࣈࣜࣥࡸࢡࢳࣥࡑࢀࡒࢀN
ᮎ➃ࡢ
calponin homology
ཬࡧࢩࢫࢸࣥࣜࢵࢳࢻ࣓ࣥࢆࡋ࡚⤖ྜࡍࡿ࠸࠺ሗ࿌ࡶ࠶ࡿ(87)ࠋࡋࡋ࡞ࡀࡽࠊࡇࡢ⤖ྜࡢヲ⣽࡞࣓࢝ࢽࢬ࣒ࡣ࡛᫂࠶ࡿࠋ୍᪉ࠊRhoGEF
ࡢ୍✀ࠊPDZ-RhoGEF
ࡶࠊࢡࢳࣥ⤖ྜࡍࡿ࠸࠺ሗ࿌ࡀ࠶ࡾࠊPDZ-RhoGEF ࢡࢳࣥࡢ⤖ྜࡼࡾࠊ
PDZ-RhoGEF
ࡢάᛶࡀᢚไࡉࢀࡿ (88,89)ࠋࡇࢀࡽࡢ」ᩘࡢRhoGEF
ࡀࢡࢳࣥ⤖ྜࡋࠊࡲࡓࡑࡢ⤖ྜࢆࡋ࡚
RhoGEF
ࡢάᛶࡀᢚไࡉࢀࡿ࠸࠺▱ぢࡣࠊᮏ◊✲࡛ぢฟࡋࡓ64
FLJ00018
ࡢάᛶᢚไᶵᵓఝ࡚࠾ࡾࠊᮏ◊✲ࢆᨭᣢࡍࡿࡶࡢ࡛࠶ࡿࠋ
PDZ-RhoGEF
ࢡࢳࣥࡢ⤖ྜ࠾࠸࡚ࡣࠊ⤖ྜ㛵ࢃࡿヲ⣽࡞࣑ࣀ㓟㓄ิࡀ᫂ࡽࡉࢀ࡚࠾ࡾࠊ
RhoGEF
୰ࡢL/IIxxFE
࠸࠺㓄ิࡀࢡࢳࣥࡢ⤖ྜ㔜せ࡛࠶ࡿࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿ
(89)ࠋFLJ00018
㓄ิ୰ࡣࠊ662-667␒┠ࡢ㓄ิ࠾࠸࡚ࠊࡇࢀఝࡓIxxxFE
࠸࠺㓄ิᏑᅾࡍࡿࡇࡽࠊᅇぢฟࡋࡓ
2
ࡘࡢࢡࢳࣥ⤖ྜ㡿ᇦࡢෆࠊC ᮎ➃ഃࡢࢡࢳࣥ⤖ྜ㓄ิࡣࠊࡇࡢ࣑ࣀ㓟ࡀࢃࡗ࡚࠸ࡿྍ⬟ᛶࡶ⪃࠼ࡽࢀࡿࠋࡋࡋ࡞ࡀࡽࠊᅇࠊྠᐃࡋ ࡓࡶ࠺୍ࡘࡢࢡࢳࣥ⤖ྜ㡿ᇦ࡛࠶ࡿ
150-283
␒┠ࡢ࣑ࣀ㓟㓄ิ୰ࡣL/IIxxFE
ࡣᏑᅾࡋ࡞࠸ࡇࡽࠊᮍ▱ࡢࢡࢳࣥ⤖ྜ㓄ิࡀ┦స⏝㛵ࢃࡗ࡚࠸ࡿྍ⬟ᛶࡶ࠶ࡾࠊᚋヲ⣽࡞᳨ウ ࡀᚲせ⪃࠼ࡽࢀࡓࠋ᭦ࠊᮏ◊✲࡛ぢฟࡋࡓ
FLJ00018
ࡢࢡࢳࣥ⤖ྜ㡿ᇦ࡛࠶ࡿࠊ150-283
␒┠ࡢ࣑ࣀ㓟㓄ิࡣࠊ
FLJ00018
ࡼࡃఝࡓRhoGEF
࡛࠶ࡿࠊPLEKHG1ཬࡧࠊPLEKHG3 ࡢDH
ࢻ࣓ࣥ㧗࠸┦ྠᛶࢆ♧ࡍ (60% ⛬ᗘ) ࡇࡽࠊࡇࢀࡽࡶࢡࢳࣥࡢ⤖ྜ㛵ࢃࡗ࡚࠸ࡿྍ⬟ᛶࡶ⪃࠼ࡽࢀࡓࠋ
F
Fig. 3--99 ࢡࢳࣥࡼࡿFFLJ00018ࡢࡢάᛶᢚไ࣓࢝ࢽࢬ࣒
GPCR่⃭ࡼࡗ࡚㐟㞳ࡋࡓGEJࡼࡿFLJ00018ࡢάᛶࡢᚋࠊFLJ00018ࡣࢡࢳࣥ⣽⬊㦵᱁ࡢ
ᵓ⠏ࢆᘬࡁ㉳ࡇࡍࠋࡇࢀࡼࡗ࡚⏕ᡂࡉࢀࡓF-ࢡࢳࣥࡀFLJ00018ࡢ150-283aaࢆྵࡴ㡿ᇦ⤖ྜࡋࠊ FLJ00018ࡢάᛶࢆᢚไࡍࡿࠋ
65
➨
➨ 4 ❶ FLJ00018 Four and a half LIM ࢻ࣓ࣥ 1 (FHL1) ࡢ┦
స⏝άᛶㄪ⠇
➨ 1 ⠇ ⥴ゝ
➨
3
❶࠾࠸࡚ࠊFLJ00018 ࡢ1-465
␒┠ࡢ࣑ࣀ㓟㡿ᇦ┦స⏝ࡍࡿ⺮ⓑ㉁ࢆࠊ㓝ẕtwo-hybrid
ἲࢆ⏝࠸࡚ࢫࢡ࣮ࣜࢽࣥࢢࡋࠊᚓࡽࢀࡓ┦స⏝ೃ⿵⺮ⓑ㉁ࡢෆࠊ24 ࢡ࣮ࣟࣥࡀࠊGenbank
ࢹ࣮ࢱ࣮࣋ࢫୖࡢ㑇ఏᏊ㓄ิ㧗࠸┦ྠᛶࢆ♧ࡋࡓ(>80%)ࠋᮏ❶࡛ࡣࠊ㓝ẕ
two-hybrid
ἲ࡛ᚓࡽࢀࡓ24
ࢡ࣮ࣟࣥࡢෆࠊ㠀➽⣽⬊ࢡࢳࣥࡣูࡢ⺮ⓑ㉁࡛࠶ࡿࠊFour and a half LIM domain 1 (FHL1)
╔┠ࡋࠊ┦స⏝ࡢ☜ㄆཬࡧࠊ┦స⏝ࡀᢸ࠺⏕⌮ⓗᙺࡘ࠸᳨࡚ウࢆ⾜ࡗࡓࠋ
FHL
⺮ⓑ㉁ࡣࠊ㦵᱁➽⣽⬊࡛ከࡃⓎ⌧ࡋ࡚࠾ࡾࠊmyopathy ࡢⓎ㛵ࢃࡿࡇࡀ♧၀ࡉࢀ࡚࠸ࡿ (90,91)ࠋ㦵᱁➽⣽⬊࡛ࡢാࡁࡘ࠸࡚ࡣࠊ࠸ࡃࡘࡢ◊✲ሗ࿌ࡀࡉࢀ࡚࠾ࡾࠊFHL3ࡀ
ࢡࢳࣥ⤖ྜࡋࠊD-ࢡࢳࢽࣥࢆࡋࡓࢡࢳࣥࡢ᮰ᐤࡍࡿ࠸࠺ሗ࿌ࡸࠊ
FHL1
ࡀ㦵᱁➽࠾࠸࡚࣑࢜ࢩࣥ⤖ྜ⺮ⓑ㉁┦స⏝ࡍࡿ࠸࠺ሗ࿌ࡀ࠶ࡿ (92,93)ࠋ
FHL1
ࡣࠊ㦵᱁➽௨እࡶ⬻ࡸ⫵ࠊ⫢⮚࡞࠾࠸࡚ࡶⓎ⌧ࡋ࡚࠸ࡿࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿ(94)ࠋ≉ࠊ࣐࢘ࢫࡢⓎ⏕ẁ㝵࠾࠸࡚ࠊ⬻࠾࠸࡚ FHL1
ࡢⓎ⌧ᕪࡀぢࡽࢀࠊ๓㒊࠾࠸࡚
FHL2
ࡀࠊᚋ㒊࠾࠸࡚FHL1
ࡢmRNA
ࡀከࡃⓎ⌧ࡋ࡚࠸ࡿࡇࡀศࡗ࡚࠸ࡿࡀࠊFHL1
ࣀࢵࢡ࢘ࢺ࣐࢘ࢫ࠾࠸࡚ࠊ⺮ⓑ㉁Ⓨ⌧ࡢኚࡣぢࡽࢀࡎࠊ
FHL1, FHL2
ࡢmRNA
㔞ࡢኚࡢព⩏ࡘ࠸࡚ࡣ࠶ࡲࡾࢃࡗ࡚࠸࡞࠸ (95)ࠋࡲࡓࠊFHL1 ࡣࠊSrc ࡼࡗ࡚ࠊࣜࣥ㓟ࡉࢀ
ࡓ
Crk-associated substrate (Cas)
ࡼࡗ࡚Ⓨ⌧㔞ࡀᢚไࡉࢀࠊࡇࢀࡀSrc
ࡼࡿࡀࢇ⣽⬊ࡢᾐ₶ࡢ⋓ᚓᐤࡋ࡚࠸ࡿ࠸࠺࣓࢝ࢽࢬ࣒ࡀᥦၐࡉࢀ࡚࠸ࡿ (96)ࠋࡋࡋ࡞ࡀࡽࠊ㦵᱁➽⣽⬊
௨እ࡛ࡢ
FHL1
ࡢാࡁࡘ࠸࡚ࡣࠊࡇࡢࡼ࠺ࠊᩓⓎⓗ࡞▱ぢࡋᚓࡽࢀ࡚࠾ࡽࡎࠊFHL1 ࡢാࡁࡘ࠸࡚ࡣࡲࡔ࠶ࡲࡾゎ᫂ࡀ㐍ࢇ࡛࠸࡞࠸ࠋ
ᅇ
two-hybrid
࡛⏝࠸ࡓprey
ࡢ㑇ఏᏊࡀ⬻ࡢcDNA
⏤᮶࡛࠶ࡿࡇࡽࠊFLJ00018 FHL1
ࡢ┦స⏝ࡀࠊ⬻ෆ࡛ఱࡽࡢᶵ⬟ࢆᯝࡓࡋ࡚࠸ࡿྍ⬟ᛶࡶ⪃࠼ࡽࢀࡿࠋࡲࡓࠊFHL1 ࡀࠊࡀࢇࡢᾐ₶ᛶࡢ㛵㐃ᛶࡀᣦࡉࢀ࡚࠸ࡿࡇࡽࠊFLJ00018 FHL1
ࡢ┦స⏝ࡀ66
⣽⬊ᙧែไᚚᶵᵓ࡛㔜せ࡞ാࡁࢆࡋ࡚࠸ࡿྍ⬟ᛶࡶ⪃࠼ࡽࢀࡿࠋ௨ୖࡢࡇࢆ㋃ࡲ࠼ࠊᮏ❶࡛ࡣࠊ
FHL1
FLJ00018
ࡢ┦స⏝ࡢヲ⣽ࠊཬࡧ┦స⏝ࡀᢸ࠺⏕⌮ⓗព⩏ࡢゎ᫂ࢆヨࡳࡓࠋ67
➨
➨ 2 ⠇ ᐇ㦂ᮦᩱ࠾ࡼࡧ᪉ἲ
1.
ࣉࣛࢫ࣑ࢻヨ⸆FHL1A
ཬࡧFHL1B
ࡢORF
ࡣࠊcDNA ࣛࣈ࣮ࣛࣜࢆᇶPCR
ἲࡼࡗ࡚ቑᖜࡉࡏࠊpF5A-Flag
࣋ࢡࢱ࣮⤌ࡳ㎸ࡲࢀࡓࠋFLJ00018
ࡢྛ✀Ḟᦆኚ␗యࠊpSRE.L-luciferase reporterࠊ pRL-SV40ࠊpF5A-CMV-neo-GE
1ཬࡧࠊpF5A-CMV-neo-GJ2ࡣ➨1
❶ཬࡧࠊ➨3
❶࡛㏙ࡓࣉࣛࢫ࣑ࢻྠ୍࡛࠶ࡿࠋ
2.
⣽⬊ᇵ㣴ࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥHEK293
⣽⬊ࠊNeuro-2a
⣽⬊ࡣࠊ37Υ, 5% CO
2ຍ‵Ẽᅽୗ࡛ࠊ10% FBS
ࢆῧຍࡋࡓDMEM࡛ᇵ㣴ࡋࡓࠋ୍㐣ᛶࡢࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡣ
Lipofectamine2000
ヨ⸆ࢆ⏝࠸࡚〇ရࡢ⏝ἲᚑࡗ࡚⾜ࡗࡓ (Life Technologies)ࠋSRE౫Ꮡⓗ㑇ఏᏊ㌿άᛶ ᐃࡣ
0.4
PgࡢDNA
ࢆࠊ࣒ࣀࣈࣟࢵࢺゎᯒࡣ
1
PgࡢDNA
ࢆࠊචỿ㝆ἲ࠾ࡼࡧpull-down
ࢵࢭࡣ2
PgࡢDNA
ࢆ⏝࠸ࡓࠋࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥᚋࠊ⣽⬊ࢆ37Υ࡛ 6-8
㛫ᇵ㣴ࡋࠊࡑࡢᚋࠊ⣽⬊ࢆ↓⾑Ύᇵᆅ⨨ࡁ࠼ࠊ16-18 㛫ᇵ㣴ࡋࡓࠋ
3. Serum response element
౫Ꮡⓗ㌿άᛶ (SREάᛶ) ᐃ24
✰ࣉ࣮ࣞࢺࢀࡓ⣽⬊ྛ✀Ⓨ⌧ࣉࣛࢫ࣑ࢻࡶpRL-SV40
ࢥࣥࢺ࣮࣏࣮ࣟࣝࣞࢱ࣮ࣉࣛࢫ࣑ࢻࠊ
pSRE.L-luciferase
ࣉࣛࢫ࣑ࢻࢆࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡋࡓࠋࢺࣛࣥࢫࣇ࢙ࢡ ࢩࣙࣥᚋࠊ⣽⬊ࢆị෭PBS
࡛1
ᅇὙίᚋࠊ ⣽⬊⁐ゎᾮࢆ⏝࠸࡚⣽⬊ࢆ⁐ゎࡋࡓࠋ⣽⬊⁐ゎᾮࢆ
13200 rpm, 10 min
࡛㐲ᚰᚋࠊୖΎࢆศ㞳ࡋࡓࠋࣝࢩࣇ࢙࣮ࣛࢮάᛶ ᐃࡣdual luciferase reporter assay system (Promega)
ࢆ⏝࠸࡚ࠊ〇ရࡢ⏝᪉ἲᚑࡗ࡚⾜ࡗࡓࠋ࣏࣮ࣞࢱ࣮άᛶ ࡣࢥࣥࢺ࣮ࣟࣝ࣋ࢡࢱ࣮ࡢάᛶᑐࡋ࡚ᶆ‽ࡋࠊࢹ࣮ࢱࡣᐇ㦂ࡢᖹᆒ್±S.D.ࢆ♧ࡋࡓࠋ4.
චỿ㝆6 cm dish
୍㐣ᛶࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡉࢀࡓ⣽⬊ࢆ300
Pl ࡢ⣽⬊⁐ゎᾮ (50 mMTris-HCl, pH 7.5, 100 mM NaCl, 0.1 mM EDTA, 1 mM Na
3VO
4, 0.5% Nonidet P-40,
68
phosphase inhibitor, protease inhibitor)
୰࡛⁐ゎࡋࡓࠋ⁐ゎᾮࢆ13200 rpm, 10 min
࡛㐲ᚰᚋࠊୖΎࡢ୍㒊ࢆศࡅࡾࠊṧࡾࡢୖΎ
1.0
PgࡢAnti-Myc
ᢠయࡲࡓࡣ1.0
PgࡢAnti-Flag
ᢠయࢆຍ࠼ࠊ
4Υ࡛ 2
㛫࣮ࣟࢸ࣮ࢩࣙࣥࢆ⾜ࡗࡓࠋࡑࡢᚋprotein G-agarose
ࢆຍ࠼ࠊ4Υ࡛ 1
㛫ࠊ࣮ࣟࢸ࣮ࢩࣙࣥࢆ⾜ࡗࡓࠋࣅ࣮ࢬࢆ
wash buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.1 mM EDTA, 1 mM Na
3VO
4, 0.1% Nonidet P-40, phosphase inhibitor solution, protease inhibitor)࡛ 3
ᅇὙίᚋࠊࣅ࣮ࢬ⤖ྜࡋࡓ⺮ⓑ㉁ࢆsample buffer
୰࡛⁐ฟࡋࡓࠋ୍ᐃ㔞ࡢචỿ㝆≀ࡣ
SDS-PAGE
࡛ศ㞳ࡉࢀࠊ⺮ⓑ㉁ࡣ࣒ࣀࣈࣟࢵࢺἲࢆ⏝࠸᳨࡚ฟࡋࡓࠋ55.
⣽⬊චᰁⰍ⣽⬊ᙧែࡢほᐹ࢝ࣂ࣮࢞ࣛࢫୖ࡛ࠊ⺯ග⺮ⓑ㉁
pFN21A-mAG-FLJ00018
ࢆྵࡴ⺮ⓑ㉁ࢆ୍㐣ᛶᙉไⓎ⌧ࡉࡏࡓ⣽⬊ࢆị෭
PBS
୍࡛ᅇὙίᚋࠊ30
ศ㛫ࠊ4% ࣃ࣒ࣛ࣍ࣝࣝࢹࣄࢻᅛᐃࡉࡏࡓࠋᅛᐃࡋ ࡓ⣽⬊0.1% Triton-X
ฎ⌮ࢆ⾜࠸ࠊPBS
࡛4
ᅇὙίᚋࠊ10% goat serum
࡛1
㛫ࣈࣟࢵ࢟ࣥࢢࡋࡓࠋࡑࡢᚋࠊAnti-Flagᢠయ࡛
1
㛫ࣛ࣋ࣝࡋࠊࡉࡽࠊᢠ࣐࢘ࢫIgG-Alexa-Flour568
࡛ ᰁⰍࡋࡓࠋᰁⰍࡋࡓ࢝ࣂ࣮࢞ࣛࢫࡣࠊPerma Fluor ࡛ฎ⌮ࡉࡏࠊ⺯ග࣓࣮ࢪࢆ⺯ග㢧ᚤ㙾ࡼࡾほᐹࡋࡓ (BZ-9000; KEYENCE)ࠋ
69