Rho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

Title

Rho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.(Doctoral

Degree)

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。

R

Rhoࣇ࢓࣑࣮ࣜపศᏊ㔞G⺮ⓑ㉁≉␗ⓗࢢ࢔ࢽࣥࢾࢡࣞ࢜ࢳࢻ஺᥮ᅉᏊ

PLEKHG2/FLJ00018ࡢ᪂つάᛶไᚚᶵᵓ࡜ࡑࡢ⣽⬊ᶵ⬟

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

2014

┠

┠ḟ

ᗎㄽ 1 ➨1 ❶ FLJ00018 ࡢ EGF ཷᐜయ่⃭ࢆ௓ࡋࡓࣜࣥ㓟໬ཬࡧάᛶ໬ ➨1 ⠇ ⥴ゝ 7 ➨2 ⠇ ᐇ㦂ᮦᩱཬࡧᐇ㦂᪉ἲ 1. ࣉࣛࢫ࣑ࢻ࡜ヨ⸆ 9 2. ⣽⬊ᇵ㣴࡜ࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥ 10

3. Serum response element ౫Ꮡⓗ㌿෗άᛶ (SRE άᛶ)  ᐃ 10

4. ࣇ࢛ࢫࣇ࢓ࢱ࣮ࢮ࡟ࡼࡿ FLJ00018 ࡢ⬺ࣜࣥ㓟໬ 10 5. ࢖࣒ࣀࣈࣟࢵࢺゎᯒ 11 6. Mn2+_{Phos-tag SDS-PAGE 11} 7. ⺯ග⺮ⓑ㉁࡟ࡼࡿ⣽⬊ᙧែࡢほᐹ 12 8. ⤫ィ࡜ศᯒ 12 ➨3 ⠇ ⤖ᯝ  1. FLJ00018 ࡢE1࢔ࢻࣞࢼࣜࣥཷᐜయ่⃭ࢆ௓ࡋࡓࢩࢢࢼࣝ࡟ࡼࡿไᚚ 13

2. FLJ00018 ࡢE1AR ࢆ௓ࡍࡿ EGF ཷᐜయ transactivation ᶵᵓ࡟ࡼࡿάᛶ໬ 14

3. FLJ00018 ࡢ EGF ่⃭࡟ࡼࡿࣜࣥ㓟໬࡜άᛶ໬ 17 4. FLJ00018 ࡢ H-Ras ࢆ௓ࡍࡿάᛶ໬ 20 5. FLJ00018 ࡣ H-Ras/MAPK ⤒㊰ࢆ௓ࡍࡿάᛶ໬ 21 6. EGF ่⃭ࢆ௓ࡋࡓ FLJ00018 ࣜࣥ㓟໬࡟㛵ࢃࡿᵓ㐀ࡢྠᐃ 23 7. EGF ่⃭࡟ࡼࡿ FLJ00018 ࣜࣥ㓟໬࡟㛵ࢃࡿ࢔࣑ࣀ㓟ࡢྠᐃ 25 8. EGF ่⃭ࢆ௓ࡋࡓ FLJ00018 ࡟ࡼࡿ⚄⤒✺㉳ᙧᡂ࡜⣽⬊ఙᒎ 27 ➨4 ⠇ ⪃ᐹ 30

➨ ➨2 ❶ 㠀ཷᐜయᆺࢳࣟࢩࣥ࢟ࢼ࣮ࢮ Src ࢆ௓ࡋࡓࢳࣟࢩࣥࣜࣥ㓟໬ ➨1 ⠇ ⥴ゝ 34 ➨2 ⠇ ᐇ㦂ᮦᩱཬࡧᐇ㦂᪉ἲ 1. ࣉࣛࢫ࣑ࢻ࡜ヨ⸆ 36 2. ⣽⬊ᇵ㣴࡜ࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥ 36 3. SH2 ࢻ࣓࢖ࣥ࢔ࣞ࢖ 36 4. ࢖࣒ࣀࣈࣟࢵࢺゎᯒ 37 5. ච␿ỿ㝆 37 ➨3 ⠇ ⤖ᯝ  1. FLJ00018 ࡢ cSrcCA ࡟ࡼࡿࢳࣟࢩࣥࣜࣥ㓟໬ 39 2. cSrcCA ࡟ࡼࡿ FLJ00018 ࡢࢳࣟࢩࣥࣜࣥ㓟໬㒊఩ࡢྠᐃ 39 3. FLJ00018 ࡢ 489 ␒┠ࡢࣜࣥ㓟໬ࢳࣟࢩࣥṧᇶ࡜≉␗ⓗ࡟ ┦஫స⏝ࡍࡿ⏕యศᏊࡢ᥈⣴ 41 4. FLJ00018 ࡜ ABL1 ࠾ࡼࡧ PIK3R3 ࡜ࡢ┦஫స⏝ 42 5. Abl1 ཬࡧ SrcCA ࡟ࡼࡿ FLJ00018 ࡢࢳࣟࢩࣥࣜࣥ㓟໬࡜ࢩࢢࢼࣝఏ㐩⤒㊰ 42 6. EphB2/Src ࢩࢢࢼࣝ⤒㊰࡟ࡼࡿ FLJ00018 ࡢࢳࣟࢩࣥࣜࣥ㓟໬ 43 ➨4 ⠇ ⪃ᐹ 46 ➨3 ❶ FLJ00018 ࡜㠀➽⣽⬊࢔ࢡࢳࣥࡢ┦஫స⏝࡜άᛶㄪ⠇ ➨1 ⠇ ⥴ゝ 48 ➨2 ⠇ ᐇ㦂ᮦᩱཬࡧᐇ㦂᪉ἲ 1. ࣉࣛࢫ࣑ࢻ࡜ヨ⸆ 50 2. 㓝ẕ two-hybrid ࢫࢡ࣮ࣜࢽࣥࢢ 50 3. ⣽⬊ᇵ㣴࡜ࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥ 51

4. Serum response element ౫Ꮡⓗ㌿෗άᛶ (SRE άᛶ)  ᐃ 51

5. ච␿ỿ㝆 51

7. ⣽⬊ච␿ᰁⰍ࡜⣽⬊ᙧែࡢほᐹ 52 ➨3 ⠇ ⤖ᯝ 1. FLJ00018 ┦஫స⏝ศᏊ࡜ࡋ࡚ࡢ㠀➽⣽⬊࢔ࢡࢳࣥࡢྠᐃ 53 2. ᇵ㣴⣽⬊ෆ࡟࠾ࡅࡿ FLJ00018 ࡜J-࢔ࢡࢳࣥཬࡧE-࢔ࢡࢳࣥ࡜ࡢ┦஫స⏝ 54 3. E-࢔ࢡࢳࣥ࡜ࡢ┦஫స⏝࡟㛵ࢃࡿ FLJ00018 ୖࡢ㓄ิࡢྠᐃ 55 4. E-࢔ࢡࢳࣥ࠾ࡼࡧJ-࢔ࢡࢳࣥ࡟ࡼࡿ FLJ00018 ࢆ௓ࡋࡓ SRE ౫Ꮡⓗ㌿෗άᛶࡢᢚไ      57 5. E-࢔ࢡࢳࣥࡀ FLJ00018 ࡜ GEJ࡜ࡢ┦஫స⏝࡟୚࠼ࡿᙳ㡪 60 6. FLJ00018 ࡜࢔ࢡࢳࣥࡢ⣽⬊ෆ࡟࠾ࡅࡿᒁᅾ 60 ➨4 ⠇ ⪃ᐹ 63 ➨

➨4 ❶ FLJ00018 ࡜ Four and a half LIM ࢻ࣓࢖ࣥ 1 (FHL1) ࡜ࡢ ┦஫స⏝࡜άᛶㄪ⠇

➨1 ⠇ ⥴ゝ 65

➨2 ⠇ ᐇ㦂ᮦᩱཬࡧᐇ㦂᪉ἲ

1. ࣉࣛࢫ࣑ࢻ࡜ヨ⸆ 67

2. ⣽⬊ᇵ㣴࡜ࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥ 67

3. Serum response element ౫Ꮡⓗ㌿෗άᛶ (SRE άᛶ)  ᐃ 67

4. ච␿ỿ㝆 67

5. ⣽⬊ච␿ᰁⰍ࡜⣽⬊ᙧែࡢほᐹ 68

➨3 ⠇ ⤖ᯝ

1. FLJ00018 ┦஫స⏝ศᏊ࡜ࡋ࡚ࡢ Four and a half LIM domain1 (FHL1)

ࡢྠᐃ 69

2. ᇵ㣴⣽⬊ෆ࡟࠾ࡅࡿ FLJ00018 ࡜ FHL1 ࡜ࡢ┦஫స⏝ 69

3. FHL1 ࡜ࡢ┦஫స⏝࡟㛵ࢃࡿ FLJ00018 ୖࡢ㓄ิ 71

4. FHL1 ࡜ࡢ┦஫స⏝࡟㛵ࢃࡿ FLJ00018 P2 ୖࡢ㓄ิࡢྠᐃ 71

6. FHL1 ࡀ FLJ00018 ࡜ GEJࡢ┦஫స⏝࡟୚࠼ࡿᙳ㡪   75 7. FHL1 ࡟ࡼࡿ FLJ00018 ࢆ௓ࡋࡓ SRE άᛶୖ᪼࡟ᑐࡍࡿ࢔ࢡࢳࣥࡢᙳ㡪 76 8. FLJ00018 ࡜ FHL1 ࡢ⣽⬊ෆ࡟࠾ࡅࡿᒁᅾཬࡧ⣽⬊ᙧែኚ໬ 77 ➨4 ⠇ ⪃ᐹ 82 ⥲ᣓ 85 ㅰ㎡ 89 ཧ⪃ᩥ⊩ 89 ␎ㄒ୍ぴ⾲ 98 ㄽᩥ┠㘓    101

1

ᗎ

ᗎㄽ

ࣄࢺࡢ࠿ࡽࡔࡣ⣙60 ඙ಶࡢ⣽⬊࠿ࡽᵓᡂࡉࢀ࡚࠾ࡾࠊࡑࢀࡒࢀࡢ⣽⬊ࡣࠊࡑࡢᛶ㉁࡟ᛂࡌ࡚ࠊ ୖ⓶⤌⧊ࠊ⤖ྜ⤌⧊ࠊ➽⤌⧊ࠊ⚄⤒⤌⧊࡞࡝ࡢ⤌⧊ࢆᵓᡂࡍࡿࠋ⤌⧊ࡀ❧యⓗࠊ୕ḟඖⓗ࡟㓄⨨ ࡉࢀྛ✀ࡢჾᐁࠊ⮚ჾࡀᙧᡂࡉࢀࠊ࠿ࡽࡔࡀࡘࡃࡽࢀ࡚࠸ࡿࠋᡃࠎࡢ࠿ࡽࡔࡣࠊእⓗ⎔ቃࡢኚ໬ ࡟㐺ᛂࡋࠊయෆࡢ⎔ቃࢆ⥔ᣢࡍࡿᜏᖖᛶࢆಖࡘࡼ࠺࡞ᶵ⬟ࢆ᭷ࡋ࡚࠸ࡿࠋᜏᖖᛶࢆ⥔ᣢࡍࡿࡓࡵ ࡟ࠊእ⏺ࡢኚ໬࡟ᑐࡋࠊ⚄⤒ఏ㐩≀㉁ࠊ࣍ࣝࣔࣥࠊࢧ࢖ࢺ࢝࢖ࣥ࡞࡝ࡢᵝࠎ࡞⏕యෆ≀㉁ࡢ㔞ⓗ ኚ໬ࡀ㉳ࡇࡾࠊ⚄⤒⣔ࠊෆศἪ⣔ࠊච␿⣔࡞࡝ࡢྛ✀ࢩࢫࢸ࣒ࡀࡑࢀ࡟ᑐᛂࡋࠊ✀ࠎࡢ⏕య཯ᛂ ࢆᘬࡁ㉳ࡇࡍࠋࡇࡢࡼ࠺࡞ᜏᖖᛶࡢ⥔ᣢࡀᔂࢀࡿ࡜㌟యࡣṇᖖ࡟ᶵ⬟ࢆಖࡘࡇ࡜ࡀ࡛ࡁࡎࠊ⑌ᝈ ࢆⓎ⑕ࡍࡿཎᅉ࡜࡞ࡿࠋ  ⣽⬊࡛ࣞ࣋ࣝࡣࠊ⣽⬊⭷ୖཬࡧ⣽⬊ෆ࡟⣽⬊እࢩࢢࢼࣝࢆཷࡅྲྀࡿᙺ┠ࢆᯝࡓࡍྛ✀ཷᐜయࡀ Ꮡᅾࡍࡿࠋཷᐜయࡀ⣽⬊እࢩࢢࢼࣝࢆ⣽⬊ෆࢩࢢࢼࣝ࡬࡜ኚ᥮ࡋࠊṇࡋ࠸⤒㊰ࢆ⤒࡚ࢩࢢࢼࣝࢆ ఏ࠼ࡿࡇ࡜࡛⣽⬊ቑṪࠊ⣽⬊ศ໬ࠊ⣽⬊㐟㉮ࠊ⣽⬊ᙧែኚ໬࡞࡝ࡢ⣽⬊ᛂ⟅ࡀᘬࡁ㉳ࡇࡉࢀࡿࠋ ⣽⬊࡟࠾ࡅࡿࢩࢢࢼࣝఏ㐩࡟㛵ࢃࡿศᏊࡢᶵ⬟࡟␗ᖖࡀ㉳ࡁࡿ࡜ᜏᖖᛶࡢ⥔ᣢࡀᔂࢀࠊ⣽⬊ࡣṇ ᖖ࡟ᶵ⬟ࢆಖࡘࡇ࡜ࡀ࡛ࡁ࡞ࡃ࡞ࡿࠋࡑࡋ࡚ࠊ⣽⬊Ṛࡸ␗ᖖ࡞⣽⬊ቑṪࢆᘬࡁ㉳ࡇࡋࠊࡑࢀࡽࡀ ಶయ࡛ࣞ࣋ࣝࡢ⑌ᝈࢆⓎ⑕ࡍࡿཎᅉ࡜࡞ࡿ࡜⪃࠼ࡽࢀࡿࠋ ࢩࢢࢼࣝఏ㐩ࡢ␗ᖖ࡟ࡼࡗ࡚ᘬࡁ㉳ࡇࡉࢀࡿ⑌ᝈࡢ୰࡛ࡶࠊࡀࢇࡣࠊ⌧ᅾࡢ᪥ᮏேࡢṚᅉ࡟༨ ࡵࡿ๭ྜࡀ᭱ࡶ㧗ࡃࠊࡑࡢඞ᭹ࡣ≉࡟ᅜᐙⓗ㔜せㄢ㢟ࡢࡦ࡜ࡘ࡛࠶ࡿࠋࡀࢇࡣᵝࠎ࡞せᅉ࡟ࡼࡾ 㑇ఏᏊኚ␗ࡀ⵳✚ࡍࡿࡇ࡜࡛ࠊᘬࡁ㉳ࡇࡉࢀࡿࠋኚ␗ࡢ✀㢮ࡣ2 ࡘ࡟኱ูࡉࢀࠊ1 ࡘࡣࠊ⣽⬊ቑ Ṫࡢࢩࢢࢼࣝఏ㐩࡟㛵ࢃࡿཎࡀࢇ㑇ఏᏊࡢgain of function ኚ␗࡟ࡼࡗ࡚ࠊᜏᖖⓗάᛶ໬ᆺ࡟࡞ ࡿࡇ࡜࡟ࡼࡿࡶࡢ࡛࠶ࡿࠋࡶ࠺୍ࡘࡣࠊṇᖖ⣽⬊࡛ࡣࠊ⣽⬊ࡢ␗ᖖ࡞ቑṪࢆᢚไࡍࡿࡀࢇᢚไ㑇 ఏᏊࡢloss of function ኚ␗࡟ࡼࡿࡶࡢ࡛࠶ࡿࠋ⺮ⓑ㉁ᵓ㐀ࡢ⣽⬊ෆࢻ࣓࢖ࣥ࡟࠾࠸࡚ࢳࣟࢩࣥ ࢟ࢼ࣮ࢮࢻ࣓࢖ࣥࢆ᭷ࡍࡿཷᐜయᆺࢳࣟࢩࣥ࢟ࢼ࣮ࢮ (RTK) ࡢከࡃࡣࠊ⭷୍ᅇ㈏㏻ᆺࡢཷᐜయ ࡛࠶ࡾᵝࠎ࡞⤌⧊ࡢቑṪ࡟㛵ࢃࡗ࡚࠸ࡿࠋୖ⓶ቑṪᅉᏊཷᐜయ (EGFR) ࡶࠊRTK ࡢ୍✀࡛࠶ࡾࠊ ࡇࡢཷᐜయࡢኚ␗࡟ࡼࡿࢩࢢࢼࣝఏ㐩ࡢ␗ᖖࡣࡀࢇࡢ୍ࡘࡢཎᅉ࡜࡞ࡾᚓࡿࠋ≉࡟ࠊ㠀ᑠ⬊ᛶ⫵ ⒴⑓ኚ࡟࠾࠸࡚EGFR ࡟」ᩘࡢኚ␗ࡀ⏕ࡌ࡚࠾ࡾࠊࡑࡢ୰࡟ࡣࠊࢳࣟࢩࣥ࢟ࢼ࣮ࢮࡀᜏᖖⓗ࡟ άᛶ໬ࡋࡓgain of function ᆺࡢኚ␗ࡶྵࡲࢀࡿ (1-3)ࠋ

2 ⏕య⭷ୖ࡟Ꮡᅾࡍࡿูࡢ኱ࡁ࡞ཷᐜయࣇ࢓࣑࣮࡛ࣜ࠶ࡿ୕㔞యࢢ࢔ࢽࣥࢾࢡࣞ࢜ࢳࢻ⤖ྜ⺮ ⓑ㉁ඹᙺᆺཷᐜయ (GPCR) ࡣࠊࣄࢺࢤࣀ࣒ࡢ⣙ 2%ࢆ༨ࡵࠊ800 ✀㢮௨ୖᏑᅾࡋ࡚࠸ࡿ࡜⪃࠼ ࡽࢀ࡚࠾ࡾࠊࢩࢢࢼࣝఏ㐩࡟㛵ࢃࡿ◊✲࡟࠾࠸࡚ࠊ⣽⬊⭷ୖࡢᶆⓗศᏊ࡜ࡋ࡚ࡣ୍␒኱ࡁ࡞ࣇ࢓ ࣑࣮࡛ࣜ࠶ࡿࠋGPCR ୖࡢኚ␗ࡶᵝࠎ࡞ࡀࢇࡢⓎ㐩࡜ࡢ㛵㐃ࡀᣦ᦬ࡉࢀ࡚࠸ࡿ (4)ࠋGPCR ࡣࠊ ⣽⬊ෆ࡟࠾࠸࡚ࠊࢢ࢔ࢽࣥࢾࢡࣞ࢜ࢳࢻ⤖ྜ⺮ⓑ㉁ (G ⺮ⓑ㉁) ࡢ୍✀࡛࠶ࡿ୕㔞య G ⺮ⓑ㉁࡜ ඹᙺࡋ࡚࠾ࡾࠊཷᐜయ่⃭࡟ࡼࡾࠊGDࢧࣈࣘࢽࢵࢺ࡜ GEJࢧࣈࣘࢽࢵࢺ࡟ゎ㞳ࡋࠊࡑࢀࡒࢀࡀ ୗὶࡢ࢚ࣇ࢙ࢡࢱ࣮࡟స⏝ࡍࡿࡇ࡜࡛ࢩࢢࢼࣝࢆఏ࠼ࡿ (5)ࠋGDࢧࣈࣘࢽࢵࢺ࡟ࡣࠊGs, Gi, Gq/11, G12/13 ࡢ 4 ࡘࡢࢧࣈࣇ࢓࣑࣮ࣜࡀᏑᅾࡋࠊ඲㒊࡛ 20 ✀⛬ࡢ㑇ఏᏊ⏘≀ࡀᏑᅾࡍࡿࠋGE, GJࡣࡑࢀࡒࢀࠊ5 ✀ཬࡧ 14 ✀ࡢ㑇ఏᏊ⏘≀ࡀᏑᅾࡋࠊࡑࢀࡒࢀࡀ␗࡞ࡿ࢚ࣇ࢙ࢡࢱ࣮࡟స⏝ࡋ ࡚࠸ࡿ (Fig. 1)ࠋGDࢧࣈࣘࢽࢵࢺࡣ୙άᛶ໬≧ែ࡛ࡣ GDP ࡜ࠊάᛶ໬≧ែ࡛ࡣ GTP ࡜⤖ྜࡋ ࡚࠾ࡾࠊGPCR ่⃭࡟ࡼࡿ GPCR ࡢᵓ㐀ኚ໬࡟క࠸ࠊGDP ࠿ࡽ GTP ࡬ࡢኚ᥮ࡀ⾜ࢃࢀࡿࠋ  ྛ✀ཷᐜయ࡟࠾ࡅࡿኚ␗࡜ࡀࢇⓎ⑕࡜ࡢ㛵ࢃࡾࡶྵࡵࠊୖ㏙ࡋࡓࡼ࠺࡟ࠊ⣽⬊ෆࢩࢢࢼࣝศᏊ ࡢ␗ᖖࢆక࠺ṇᖖ࡞ࢩࢢࢼࣝఏ㐩ࡢ◚⥢࡟ࡼࡿࡀࢇࡢⓎ⑕࡟࠾࠸࡚ࠊ㑇ఏᏊኚ␗ࡀከẁ㝵࡟⵳✚ ࡍࡿ࡜⪃࠼ࡽࢀ࡚࠸ࡿࠋ኱⭠⒴ࡢሙྜࠊึࡵ࡟ࠊ㑇ఏⓗせᅉࡸࠊእⓗ⎔ቃ࡟⏤᮶ࡍࡿ࢖ࢽࢩ࢚࣮ Fig. 1 ୕୕㔞యG ⺮⺮ⓑ㉁ࡢ✀㢮࡜࢚ࣇ࢙ࢡࢱ࣮⺮ⓑ㉁ࡢάᛶ໬

3 ࢱ࣮ࡀࠊࡀࢇᢚไ㑇ఏᏊࡢAPC ࡢḞᦆࢆᘬࡁ㉳ࡇࡍࠋ⥆࠸࡚ࠊ⣽⬊ቑṪ࡟㛵ࢃࡿཎࡀࢇ㑇ఏᏊ ࡛࠶ࡿK-Ras ୖ࡟άᛶ໬ᆺኚ␗ࡀ⏕ࡌࡿࠋᐇ㝿ࠊ኱⭠ࡢṇᖖୖ⓶ࡀ␗ᖖቑṪࡋࡓึᮇࡢ⭢⭘࡟ ࠾࠸࡚ࠊ࠶ࡿ୍ᐃࡢ☜⋡࡛APC ኚ␗ࡀ⏕ࡌ࡚࠸ࡿࡇ࡜ࡀ♧၀ࡉࢀ࡚࠾ࡾࠊ⭢⭘ࡢ኱ࡁࡉ࡜ K-Ras ࡢኚ␗࡟࠾ࡅࡿ┦㛵㛵ಀࡀᣦ᦬ࡉࢀ࡚࠸ࡿࠋ᭦࡟ࡇࡢᚋࠊࡀࢇᢚไ㑇ఏᏊ࡛࠶ࡿ p53 ࡢኚ␗࡟ ࡼࡾࠊᾐ₶ᛶ⒴ࡀⓎ㐩ࡍࡿ࡜⪃࠼ࡽࢀ࡚࠸ࡿ (Fig. 2) (6)ࠋ

K-Ras ࡣ Ras ࢫ࣮ࣃ࣮ࣇ࢓࣑࣮ࣜ࡟ᒓࡍࡿపศᏊ㔞 GTP ⤖ྜ⺮ⓑ㉁ (small GTPase) ࡢ୍ ✀࡛࠶ࡿࠋsmall GTPase ࡣ༢㔞య࡛ാࡁࠊ⤖ྜࡍࡿ GTP ࡜ GDP ࡢ⤖ྜ≧ែ࡟ࡼࡗ࡚άᛶ໬

≧ែࡀኚ໬ࡋࠊ୙άᛶ໬≧ែ࡛ࡣ GDP ࡜ࠊάᛶ໬≧ែ࡛ࡣ GTP ࡜⤖ྜࡋ࡚࠾ࡾࠊGDP/GTP

஺᥮཯ᛂࡣࢢ࢔ࢽࣥࢾࢡࣞ࢜ࢳࢻ஺᥮ᅉᏊ (GEF)ࠊGTPase άᛶ໬⺮ⓑ㉁ (GAP)ࠊࢢ࢔ࢽࣥࢾ ࢡࣞ࢜ࢳࢻゎ㞳ᢚไᅉᏊ (GDI) ࡞࡝࡟ࡼࡗ࡚ไᚚࡉࢀࡿࠋGEF ࡣ GDP ࠿ࡽ GTP ࡬ࡢኚ᥮ࢆࠊ GAP ࡣ Small GTPase ⮬㌟ࡢ GTP ࢆ GDP ࡬ศゎࡍࡿ GTPase άᛶࢆάᛶ໬ࡉࡏࠊGDI ࡣ GDP

⤖ྜᆺࡢGTPase ࢆᏳᐃ໬ࡉࡏࡿᙺ๭ࢆᯝࡓࡋ࡚࠸ࡿ (Fig. 3)ࠋRas ࢫ࣮ࣃ࣮ࣇ࢓࣑࣮ࣜ࡟ᒓࡍ

ࡿsmall GTPase ࡣࠊ㑇ఏᏊᵓ㐀࠿ࡽࠊRas, Rho, Rab, Ran, Arf ࡢ 5 ࡘࡢࢧࣈࣇ࢓࣑࣮ࣜ࡟ศ

㢮ࡉࢀࠊ⣽⬊ᶵ⬟ࡶࡑࢀࡒࢀࡢᵓ㐀࠿ࡽ኱ู࡛ࡁࡿࠋࡑࢀࡒࢀ⣽⬊ᶵ⬟࡟ࡘ࠸࡚ࡣࠊRas ࡣ⣽⬊

ቑṪࠊRho ࡣ࢔ࢡࢳࣥ⣽⬊㦵᱁෌ᵓ⠏ࢆ௓ࡋ࡚⣽⬊ᙧែࡸ㐠ືᛶࡢไᚚࢆᢸ࠸ࠊRab ࡣ᰾ෆ㍺

㏦ࠊࡑࡋ࡚Ran ཬࡧ Arf ࡣࠊ⣽⬊ෆᑠ⬊㍺㏦࡟㛵୚ࡍࡿ (Fig. 4)ࠋ

4

㏆ᖺࠊGPCR ࢩࢢࢼࣝ࡟ࡼࡿ Small GTPase ࢩࢢࢼࣝࡢไᚚᶵᵓࡀ᫂ࡽ࠿࡟࡞ࡾࡘࡘ࠶ࡿ (Fig. 5)ࠋ≉࡟ࠊ⣽⬊ᙧែࡸ㐠ືᛶࡢไᚚ࡟㔜せ࡞࢔ࢡࢳࣥ⣽⬊㦵᱁ࡢ෌⦅ᡂࢆᢸ࠺ Rho ࣇ࢓࣑ ࣮ࣜSmall GTPase (RhoGTPase) ࡢኚ␗ࡣ⒴ࡢᾐ₶ࠊ㌿⛣⬟ࡢ⋓ᚓ࡟㔜せ࡛࠶ࡿ࡜⪃࠼ࡽࢀ࡚ ࠾ࡾࠊGPCR ࡟ࡼࡿ RhoGTPase ࡢάᛶไᚚᶵᵓࢆ⌮ゎࡍࡿࡇ࡜ࡣࡀࢇࡢ⑓ែゎ᫂࡟㔜せ࡛࠶

ࡿ࡜⪃࠼ࡽࢀ࡚࠾ࡾࠊ᭱㏆ࠊࡀࢇ⑓ኚ࡟࠾࠸࡚ࠊRac ࡢኚ␗ࡀⓎぢࡉࢀ࡚࠸ࡿ (7)ࠋRhoGTPase

ࡣࠊࣄࢺ࡛ࡣ23 ✀ࡀ▱ࡽࢀ࡚࠾ࡾࠊRhoA, Rac1, Cdc42 ࡣ≉࡟◊✲ࡀ㐍ࢇ࡛࠸ࡿศᏊ࡛࠶ࡿ (8)ࠋ

RhoA ࡀάᛶ໬ࡍࡿ࡜࢔ࢡࢳࣥࢫࢺࣞࢫࣇ࢓࢖ࣂ࣮ࡀᙧᡂࡉࢀࠊ⣽⬊ࡢ཰⦰ࠊ᥋╔࡟ᐤ୚ࡋ࡚࠸

Fig. 33 SSmall GTPase ࢩࢢࢼࣝศᏊࡢࢩࢢࢼࣝࢫ࢖ࢵࢳ

5

ࡿࠋࡲࡓࠊRac1 ࡢάᛶ໬ࡣࠊⴥ≧௬㊊ (lamellipodia) ࢆࠊCdc42 ࡢάᛶ໬࡛ࡣ⣒≧௬㊊ (filopodia) ࡢᙧᡂࡀࡑࢀࡒࢀಁ㐍ࡉࢀࠊ⣽⬊㉁ࢆᗈࡆࠊ⣽⬊ࡢఙᒎࡸࠊ⚄⤒✺㉳ࡢఙ㛗࡞࡝࡟ 㛵୚ࡍࡿ࡜⪃࠼ࡽࢀ࡚࠸ࡿ (9) (Fig. 6)ࠋRhoGTPase ࡢ GDP ⤖ྜ࠿ࡽ GTP ⤖ྜ࡬ࡢኚ໬࡟క ࠺άᛶ໬ࡣࠊRhoGTPase ࡟≉␗ⓗ࡞ GEF (RhoGEF) ࡟ࡼࡗ࡚ไᚚࡉࢀࡿ (10)ࠋRhoGEF ࡣ 2 ࡘࡢࣇ࢓࣑࣮ࣜ࡟ศ㢮ࡉࢀࠊ1 ࡘࡣ GEF ࡢゐ፹άᛶࢆ᭷ࡍࡿ Dbl homology domain (DH ࢻ࣓ ࢖ࣥ) ࡜⣽⬊⭷࡬ࡢ⛣⾜࡟㛵ࢃࡿ Pleckstrin homology domain (PH ࢻ࣓࢖ࣥ) ࢆ᭷ࡍࡿ Dbl ࣇ

࢓࣑࣮࡛ࣜ࠶ࡿࠋ⌧ᅾࡲ࡛࡟ࠊࣄࢺ࡟࠾࠸࡚Dbl ࣇ࢓࣑࣮ࣜࡢ RhoGEF ࡣ⣙ 70 ✀ྠᐃࡉࢀ࡚

࠸ࡿ (11,12)ࠋRhoGEF ࡢࡶ࠺ 1 ࡘࡢࣇ࢓࣑࣮ࣜࡣࠊDH ࡸ PH ࢻ࣓࢖ࣥࢆ᭷ࡉ࡞࠸௦ࢃࡾ࡟ Dock homology region (DHR) 2 ࢻ࣓࢖ࣥࢆᣢࡘ CDM-zizimin homology (CZH) ⺮ⓑ㉁ࣇ࢓࣑

࣮࡛ࣜ࠶ࡾࠊࣄࢺ࡛ࡣ11 ✀ࡀ▱ࡽࢀ࡚࠸ࡿ (13)ࠋࡇࢀࡽࡢෆࠊDbl ࣇ࢓࣑࣮ࣜࡢ RhoGEF ࡢ

୍✀࡛࠶ࡿࠊleukemia-associated RhoGEF (LARG), P115-RhoGEF, PDZ-RhoGEF ࡣࠊDH ࢻ ࣓࢖ࣥࠊPH ࢻ࣓࢖ࣥ࡟ຍ࠼ࠊRegulator of G-protein signaling domain (RGS ࢻ࣓࢖ࣥ) ࢆ᭷

ࡋ࡚࠾ࡾࠊࡇࡢRGS ࢻ࣓࢖ࣥࢆ௓ࡋ࡚୕㔞య G ⺮ⓑ㉁ࡢ GD12/13 ࡜⤖ྜࡍࡿࡇ࡜࡛άᛶ໬ࡉ

ࢀࠊRhoA ࡢ GDP/GTP ஺᥮཯ᛂࢆ௰௓ࡍࡿ (14-16)ࠋࡲࡓࠊp63-RhoGEF ࡸࠊTrio, Karilin ࡜

࠸ࡗࡓRhoGEF ࡣ୕㔞య G ⺮ⓑ㉁ࡢ Gq ࢩࢢࢼࣝ࡟ࡼࡗ࡚άᛶ໬ࡉࢀࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿ

(17-19)ࠋ୍᪉ࠊ୕㔞య G ⺮ⓑ㉁ࡢ GEJࢧࣈࣘࢽࢵࢺ࡟ࡼࡗ࡚άᛶ໬ࡉࢀࡿ RhoGEF ࡟ P-Rex1 ࡀ▱ࡽࢀ࡚࠸ࡿ (20)ࠋάᛶ໬ࡉࢀࡓ P-Rex1 ࡣ Rac ࡢάᛶ໬ࢆ௓ࡋࠊ⣽⬊ᙧែኚ໬࡟㛵ࢃࡗ࡚ ࠸ࡿ࡜⪃࠼ࡽࢀ࡚࠾ࡾࠊ㏆ᖺࠊஙࡀࢇᝈ⪅ࡢࡀࢇ⑓ኚ࡟࠾࠸࡚ࠊP-Rex1 ࡢ㐣๫Ⓨ⌧ࡀぢࡽࢀࡿ ࡇ࡜ࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿ (21)ࠋ

6 PLEKHG2/FLJ00018 ࡣࠊ࣐࢘ࢫࡢࣜࣥࣃ⭘ࡸ㦵㧊ᛶⓑ⾑⑓ࣔࢹࣝ࡟࠾࠸࡚ࠊࣞࢺࣟ࢘࢖ࣝ ࢫࡢྲྀࡾ㎸ࡳࢧ࢖ࢺ࡛࠶ࡿEvi24 ࡬ࡢࣉࣟ࢘࢖ࣝࢫྲྀࡾ㎸ࡳ࡟ࡼࡗ࡚ࠊⓎ⌧ୖ᪼ࡉࢀࡿ Dbl ࣇ ࢓࣑࣮ࣜࡢRhoGEF ࡢ୍✀࡜ࡋ࡚ྠᐃࡉࢀࡓ (22)ࠋࡲࡓࠊ௚ࡢ Dbl ࣇ࢓࣑࣮ࣜࡢ RhoGEF ࡜ ྠᵝ࡟ࠊFLJ00018 ࡶ N ᮎ➃௜㏆ࡢ㓄ิ࡟ DH ࢻ࣓࢖ࣥ࡜ PH ࢻ࣓࢖ࣥࢆᣢࡕࠊUeda ࡽ࡟ࡼ ࡾࠊFLJ00018 ࡢ DH ࢻ࣓࢖ࣥࢆྵࡴ N ᮎ➃௜㏆ࡢ㓄ิࡀ GPCR ่⃭࡟ࡼࡾ㐟㞳ࡋࡓ୕㔞య G ⺮ⓑ㉁ࡢ GEJࢧࣈࣘࢽࢵࢺ࡜┤᥋⤖ྜࡍࡿࡇ࡜࡟ࡼࡗ࡚άᛶ໬ࡉࢀࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿ (23)ࠋάᛶ໬ࡉࢀࡓ FLJ00018 ࡣࠊRhoGTPase ࡢ Rac1, Cdc42 ࢆάᛶ໬ࡋࠊ⣽⬊ᙧែࡢኚ໬ࢆ ᘬࡁ㉳ࡇࡍࠋ㏆ᖺࠊච␿⣔ࡢᇵ㣴⣽⬊ࡢ୍✀࡛࠶ࡿjurkat T ⣽⬊࡟࠾࠸࡚ࡶࠊFLJ00018 ࡀ GEJ ࡟ࡼࡗ࡚άᛶ໬ࡉࢀࠊ⣽⬊㐟㉮࡟㛵୚ࡋ࠺ࡿࡇ࡜ࡀሗ࿌ࡉࢀࡓ (24)ࠋ୍⯡ⓗ࡟ࠊRhoGEF ศᏊ ࡣ㧗ศᏊ㔞ࢆᣢࡘᕧ኱࡞⺮ⓑ㉁࡛࠶ࡾࠊ」㞧࡞άᛶไᚚࢆཷࡅࡿࡇ࡜ࡀ⪃࠼ࡽࢀࡿࠋFLJ00018 ࡶࠊ1386 ࡶࡢ࢔࣑ࣀ㓟ࢆᣢࡕࠊ」㞧࡞ไᚚᶵᵓࡀᏑᅾࡍࡿࡇ࡜ࡀ⪃࠼ࡽࢀࡿࠋࡋ࠿ࡋ࡞ࡀࡽࠊ ⌧ᅾࡢ࡜ࡇࢁࠊFLJ00018 ࡢ DH ࢻ࣓࢖ࣥࠊPH ࢻ࣓࢖ࣥ௨እࡢ㓄ิ࡟┠❧ࡗࡓᶵ⬟ࢻ࣓࢖ࣥࡣ ࡞ࡃࠊDH, PH ࢻ࣓࢖ࣥ௨እࡢṧࡾࡢ C ᮎ➃ഃࡢᵓ㐀ࡀ⣽⬊ᶵ⬟࡟୚࠼ࡿᙺ๭ࡣᮍ▱࡛࠶ࡿࠋ ࡑࡇ࡛ᮏ◊✲࡛ࡣࠊ⒴ࡢᾐ₶࣭㌿⛣ᶵᵓࡢ⑓ែࡢ୍➃ࢆゎ᫂ࡍࡿ࡭ࡃࠊFLJ00018 ࡢ᪂つάᛶไ ᚚᶵᵓࡢ᥈⣴࡜ࠊࡑࡢάᛶไᚚࡀ⣽⬊ᶵ⬟࡟୚࠼ࡿᙳ㡪ࡢ᳨ウࢆ⾜ࡗࡓࠋ

Fig. 66 RRhoGTPase ࡢ✀㢮࡜ᶵ⬟

7

➨

➨

1 ❶ FLJ00018 ࡢ EGF ཷᐜయ่⃭ࢆ௓ࡋࡓࣜࣥ㓟໬ཬࡧάᛶ໬

➨

1 ⠇ ⥴ゝ

࠸ࡃࡘ࠿ࡢRhoGEF ࡣࠊ」ᩘࡢ GPCR ࢩࢢࢼࣝ࡟ࡼࡿไᚚࢆཷࡅࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿࠋ

ᵓ㐀୰࡟RGS ࢻ࣓࢖ࣥࢆྵࡴ RhoGEF ࡛࠶ࡿࠊp115-RhoGEF ࡸ LARG, PDZ-RhoGEF ࡣࠊ

GD13 ࡜ࠊࡑࡢ RGS ࢻ࣓࢖ࣥࢆ௓ࡋ࡚άᛶ໬ࡉࢀࠊRhoA ࡟ᑐࡍࡿ GEF άᛶࢆ♧ࡍ (14)ࠋ ୍᪉࡛ࠊPDZ-RhoGEF ࡣࠊࡑࡢ N ᮎ➃ࡢᵓ㐀୰ࡢ PSD-95/Dlg/Zo-1 domain (PDZ ࢻ࣓࢖ࣥ) ࢆ ௓ࡋࠊLPA ཷᐜయ࡜⤖ྜࡋࠊRhoA ࢆάᛶ໬ࡍࡿ (25)ࠋࡲࡓࠊPDZ-RhoGEF ࢆྵࡴࡇࢀࡽࡢ RhoGEF ⩌ࡣูࡢ୕㔞య G ⺮ⓑ㉁Dࢧࣈࣘࢽࢵࢺ࡛࠶ࡿࠊGDq ࡜ࡶࠊPDZ ࢻ࣓࢖ࣥࢆ௓ࡋ࡚┦ ஫స⏝ࡋࠊRhoA ࢆάᛶ໬ࡍࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿ (26)ࠋ

⺮ⓑ㉁ࡢࣜࣥ㓟໬ࡣࠊ⣽⬊ෆࢩࢢࢼࣝఏ㐩ࢆᢸ࠺⺮ⓑ㉁ࢆάᛶ໬ࡍࡿ㔜せ࡞ᡭẁࡢ୍ࡘ࡛࠶ࡿࠋ

㏆ᖺࠊቑṪᅉᏊཷᐜయ่⃭ࡸ⚄⤒ᡂ㛗ᅉᏊཷᐜయ่⃭࡟ࡼࡗ࡚άᛶ໬ࡉࢀࡿRhoGEF ࡢᏑᅾࡶ

ᗈࡃ▱ࡽࢀ࡚ࡁ࡚࠾ࡾࠊ⣙70 ✀ࡢ RhoGEF ࡢ࠺ࡕࠊᑡ࡞ࡃ࡜ࡶ 19 ✀ࡣቑṪᅉᏊཷᐜయ࡟ࡼࡗ

࡚άᛶ໬ࢆཷࡅࡿࡇ࡜ࡀ▱ࡽࢀ࡚࠸ࡿ (27)ࠋࡇࡢ୰࡟ࡣࠊEPIX ࡸ Vav3, Asef ࡜࠸ࡗࡓ RhoGEF

ࡶྵࡲࢀࡿࠋAsef ࡣࠊEGFR ࡟ࡼࡗ࡚ࢳࣟࢩࣥࣜࣥ㓟໬ࡉࢀࡿࡇ࡜࡟ࡼࡾάᛶ໬ࡉࢀࠊRac1, Cdc42 ࡢάᛶ໬ࢆᘬࡁ㉳ࡇࡍ (28)ࠋࡲࡓࠊEPIX ࡣࠊ⥺⥔ⱆ⣽⬊ቑṪᅉᏊཷᐜయ (FGFR) ࡢୗ ὶ࡟࠾࠸࡚ࢭࣜࣥ/ࢫࣞ࢜ࢽࣥࣜࣥ㓟໬ࢆཷࡅ࡚άᛶ໬ࡉࢀࡿࡇ࡜ࡀ▱ࡽࢀ࡚࠸ࡿ (29)ࠋ  ࡉࡽ࡟㏆ᖺࠊࣜࣥ㓟໬௨እࡢ⺮ⓑ㉁⩻ヂᚋಟ㣭࡟ࡼࡿ⺮ⓑ㉁ࡢᒁᅾኚ໬ࠊཬࡧάᛶไᚚࡢᶵᵓ ࡶ᫂ࡽ࠿࡟ࡉࢀࡘࡘ࠶ࡿࠋ౛࠼ࡤࠊp63-RhoGEF ࡣࠊࡑࡢ⺮ⓑ㉁ࡢ 23, 25 ཬࡧ 26 ␒┠ࡢࢩࢫ ࢸ࢖ࣥṧᇶࡀࣃ࣑ࣝࢺ࢖ࣝ໬ࡉࢀࠊࡇࢀ࡟ࡼࡾ⣽⬊⭷࡬ࡢᒁᅾࡀྍ⬟࡜࡞ࡾ RhoA άᛶࢆ♧ࡍ ࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿ (30)ࠋ ࡇࡢࡼ࠺࡟ࠊ࠸ࡃࡘ࠿ࡢRhoGEF ࡣ」ᩘࡢ GPCR ࢩࢢࢼࣝ࡟ࡼࡗ࡚άᛶ໬ࡉࢀࠊRhoGTPase ࢆάᛶ໬ࡍࡿࠋࡲࡓࠊRhoGEF ࡣ GPCR ࢆάᛶ໬ࡍࡿ୍᪉࡛ࠊGPCR ௨እࡢࢩࢢࢼࣝ࡟ࡼࡗ࡚ ࣜࣥ㓟໬ࡸࣃ࣑ࣝࢺ࢖ࣝ໬ࢆྵࡴ⩻ヂᚋಟ㣭ࢆ௓ࡋࠊάᛶ໬ࡉࢀࡿࡇ࡜ࡀ▱ࡽࢀࡘࡘ࠶ࡾࠊࡇࢀ ࡣ୍ࡘࡢRhoGEF ࡀ」ᩘࡢࢩࢢࢼࣝఏ㐩⤒㊰࡟㛵୚ࡋ࠺ࡿࡇ࡜ࢆ♧၀ࡋ࡚࠸ࡿࠋࡋ࠿ࡋ࡞ࡀࡽࠊ FLJ00018 ࡢ GEJ௨እࡢࢩࢢࢼࣝ࡟ࡼࡿάᛶ໬ᶵᵓࡢ᭷↓ࡣᮍࡔ᫂ࡽ࠿࡟ࡉࢀ࡚࠸࡞࠸ࠋࡇࡢࡼ

8

࠺࡞⫼ᬒ࠿ࡽࠊᮏ❶࡛ࡣࠊFLJ00018 ࡢ᪂つࡢάᛶ໬ᶵᵓࡢ᭷↓ࠊFLJ00018 ࡢ⩻ヂᚋಟ㣭࡟ࡼ

ࡿάᛶ໬ࡢ᭷↓࡟ࡘ࠸᳨࡚ウࢆ⾜࠸ࠊࡑࡢάᛶ໬ᶵᵓࡢヲ⣽ࢆ᫂ࡽ࠿࡟ࡍࡿࡇ࡜ࢆ┠ⓗ࡜ࡋࡓࠋ

9

➨

➨

2 ⠇ ᐇ㦂ᮦᩱ࠾ࡼࡧ᪉ἲ

1. ࣉࣛࢫ࣑ࢻ࡜ヨ⸆

pcDNA3.1-E1- ࢔ ࢻ ࣞ ࢼ ࣜ ࣥ ཷ ᐜ య (E1AR), pcDNA3.1-GE1, pcDNA3.1-GJ2, pcDNA3.1-H-Ras AC (H-Ras ᜏᖖⓗάᛶ໬ᆺኚ␗య), pcDNA3.1-H-Ras T17N (H-Ras ࢻ࣑ࢼ ࣥࢺࢿ࢞ࢸ࢕ࣈኚ␗య), pcDNA3.1-RhoA T19N, pcDNA3.1-Rac1 T17N, pcDNA3.1-Cdc42 T17N, pCMV5 ࡣࠊMissouri S&T cDNA Resource Center ࠿ࡽ㉎ධࡋࡓࠋࡇࢀࡽࡢࣉࣛࢫ࣑ࢻ ࡣࠊPCR ἲࢆ⏝࠸࡚ pF4A-CMV ࣋ࢡࢱ࣮ࠊpF5A-CMV-neo ࣋ࢡࢱ࣮ཬࡧࠊpFN21A ࣋ࢡࢱ࣮ ࡟⤌ࡳ᥮࠼ࡽࢀࡓࠋpCMV5-EARKct ࡣࠊዉⰋඛ➃኱Ꮫ㝔኱Ꮫ ఀᮾᗈඛ⏕࠿ࡽ౪୚ࡉࢀࡓࠋ pFC21A-Halo-EGFR, pFN21K-Halo-Vav3 ࡣࠊ࠿ࡎࡉ DNA ◊✲ᡤࡼࡾ౪୚ࡉࢀࡓࠋFLJ00018

㑇ఏᏊࡢ᏶඲㛗cDNA ࢡ࣮ࣟࣥࡣࠊ࠿ࡎࡉ DNA ࣄࢺ㛗㙐㑇ఏᏊゎㄞࣉࣟࢪ࢙ࢡࢺ࡟ࡼࡾᚓࡽ

ࢀࡓ (31)ࠋ㔝⏕ᆺ FLJ00018 ࡢⓎ⌧ࢡ࣮ࣟࣥࢆᚓࡿⅭ࡟ FLJ00018 ࡢ ORF 㡿ᇦࡣ Gateway ࢩ

ࢫ ࢸ ࣒ (Life Technologies) ࢆ⏝࠸࡚ pcDNA3.1-DEST47 ࣋ࢡࢱ࣮࡟⤌ࡳ᥮࠼ࡽࢀࡓࠋ

pcDNA3.1-DEST47-FLJ00018 ࡣࠊไ㝈㓝⣲ฎ⌮ࠊPCR ἲࢆ⏝࠸࡚ pFN21A-Myc ࣋ࢡࢱ࣮ࡶ

ࡋࡃࡣpFN21K-Halo ࣋ࢡࢱ࣮࡟⤌ࡳ᥮࠼ࡽࢀࡓࠋྛᅗ࡟♧ࡋࡓ FLJ00018 ࡢྛ✀Ḟᦆኚ␗య

ࡣࠊpFN21A-Myc-FLJ00018 ࢆ⏝࠸࡚ไ㝈㓝⣲ฎ⌮ࠊPCR ἲࢆ⏝࠸࡚స〇ࡉࢀࡓࠋFLJ00018 ࡢ

ྛ✀Ḟᦆኚ␗య࠾ࡼࡧࠊFLJ00018 T680A ࡣ pFN21A-Myc-FLJ00018 ࢆ㗪ᆺ࡟ࡋࠊPCR ἲ (Stratagene) ࢆ⏝࠸࡚స〇ࡋࡓࠋ࢔ࢨ࣑ࢢ࣮ࣜࣥ (AG ; Azami green) ࡣࠊ࢖ࢩࢧࣥࢦࡼࡾ༢ 㞳ࡉࢀࡓ᪂つ⺯ග⺮ⓑ㉁࡛࠶ࡿ (32)ࠋࡇࡢ⺯ග⺮ⓑ㉁ࢆ࢔࣑ࣀ㓟⨨᥮࡟ࡼࡾ༢㔞య࡛Ꮡᅾ࡛ࡁ ࡿࡼ࠺࡟ࡋࡓ⺮ⓑ㉁ࣉࣛࢫ࣑ࢻࠊphmAG1-MCLinker ࡣ MBL ࡼࡾ㉎ධࡋࡓࠋࡇࡢ㑇ఏᏊࢆ⏝ ࠸ࠊPCR ἲࠊไ㝈㓝⣲ฎ⌮࡟ࡼࡾ pFN21A-mAG-FLJ00018 ࢆస〇ࡋࡓࠋpSRE.L-luciferase reporter ࡣࠊStratagene ࠿ࡽࠊpRL-SV40 ࡣࠊNippon Gene ࠿ࡽ㉎ධࡋࡓࠋIsoproterenol ࠾ ࡼࡧKN-93 ཬࡧࠊSB202190 ࡣࠊCalbiochem, EGF ࡣࠊPeprotech ࠿ࡽ㉎ධࡋࡓࠋAG1478, U0126 ࡣࠊWako ࠿ࡽ㉎ධࡋࡓࠋSP600125 ࡣࠊSigma ࠿ࡽ㉎ධࡋࡓࠋphosphase inhibitor ࠾ࡼࡧ protease inhibitor ࡣࠊRoche ࠿ࡽ㉎ධࡋࡓࠋLambda phosphatase ࡜࢔ࣝ࢝ࣜࣇ࢛ࢫࣇ࢓ࢱ࣮ ࢮࡣࠊNew England Biolabs ࠿ࡽ㉎ධࡋࡓࠋ

10

22. ⣽⬊ᇵ㣴࡜ࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥ

NIH3T3 ⣽⬊ࡣࠊ37Υ, 5% CO2ຍ‵኱Ẽᅽୗ࡛ࠊ10% calf serum (CS) ࢆῧຍࡋࡓࢲࣝ࣋ࢵ

ࢥኚἲ࢖࣮ࢢࣝᇵᆅ (DMEM) ࡛ᇵ㣴ࡋࡓࠋHEK293 ⣽⬊࡜ Neuro-2a ⣽⬊ࡣ 37Υ, 5% CO2ຍ

‵኱Ẽᅽୗ࡛ࠊ10% Fetal Bovine Serum (FBS) ࢆῧຍࡋࡓ DMEM ࡛ᇵ㣴ࡋࡓࠋ୍㐣ᛶࡢࢺࣛ

ࣥࢫࣇ࢙ࢡࢩࣙࣥࡣ Lipofectamine-Plus ヨ⸆ࢆ⏝࠸࡚〇ရࡢ౑⏝ἲ࡟ᚑࡗ࡚⾜ࡗࡓ (Life

Technologies)ࠋserum response element (SRE) ౫Ꮡⓗ㑇ఏᏊ㌿෗άᛶ ᐃ࡟ࡣ 0.4 Pg ࡢ DNA

ࢆࠊ࢖࣒ࣀࣈࣟࢵࢺゎᯒ࡟ࡣ1 Pg ࡢ DNA ࢆࠊච␿ỿ㝆ἲ࡟ࡣ 2 Pg ࡢ DNA ࢆ⏝࠸ࡓࠋࢺࣛࣥ

ࢫࣇ࢙ࢡࢩࣙࣥࡢ3 ᫬㛫ᚋࠊ⣽⬊࡟ 1× insulin-transferrin-selenium-X (Invitrogen) ࢆຍ࠼ࠊ 6-8 ᫬㛫ᇵ㣴ࡋࡓࠋࡑࡢᚋࠊ⣽⬊ࢆ↓⾑Ύᇵᆅ࡟⨨ࡁ᥮࠼ࠊ16-18 ᫬㛫ᇵ㣴ࡋࡓࠋ

3. Serum response element ౫Ꮡⓗ㌿෗άᛶ (SRE άᛶ)  ᐃ

24 ✰ࣉ࣮ࣞࢺ࡟᧛✀ࡋࡓ⣽⬊࡟ྛ✀Ⓨ⌧ࣉࣛࢫ࣑ࢻ࡜࡜ࡶ࡟ pRL-SV40 ࢥࣥࢺ࣮࣏࣮ࣟࣝࣞ

ࢱ࣮ࣉࣛࢫ࣑ࢻࠊpSRE.L-luciferase ࣉࣛࢫ࣑ࢻࢆࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡋࡓࠋྛᅗ࡟♧ࡋࡓ᫬

㛫࡜⃰ᗘ࡛ྛ✀㜼ᐖ๣ฎ⌮ࢆ⾜ࡗࡓᚋࠊ⣽⬊ࢆ่⃭ࡋࡓࠋࡑࡢᚋࠊ⣽⬊ࢆị෭PBS ࡛ 1 ᅇὙί

ᚋࠊ ⣽⬊⁐ゎᾮࢆ⏝࠸࡚⣽⬊ࢆ⁐ゎࡋࡓࠋ⣽⬊⁐ゎᾮࢆ 13200 rpm, 10 min ࡛㐲ᚰᚋࠊୖΎࢆ ศ㞳ࡋࡓࠋࣝࢩࣇ࢙࣮ࣛࢮάᛶ ᐃࡣdual luciferase reporter assay system (Promega) ࢆ⏝ ࠸࡚ࠊ〇ရࡢ౑⏝᪉ἲ࡟ᚑࡗ࡚⾜ࡗࡓࠋ࣏࣮ࣞࢱ࣮άᛶࡣࢥࣥࢺ࣮ࣟࣝ࣋ࢡࢱ࣮ࡢάᛶ࡟ᑐࡋ࡚ ᶆ‽໬ࡋࠊࢹ࣮ࢱࡣᐇ㦂ࡢᖹᆒ್±S.D.ࢆ♧ࡋࡓࠋ

4. ࣇ࢛ࢫࣇ࢓ࢱ࣮ࢮ࡟ࡼࡿ FLJ00018 ࡢ⬺ࣜࣥ㓟໬

ࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡋࡓ⣽⬊ࢆྛᅗ࡟♧ࡋࡓ⃰ᗘ࡜᫬㛫࡟ࡼࡾ่⃭ࡋࡓࠋࡑࡢᚋࠊ⣽⬊ࢆị ෭PBS ୍࡛ᅇὙίࡋࠊ⣽⬊⁐ゎᾮ (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.5% Nonidet P-40, protease inhibitor) ୰࡛⁐ゎࡋࠊ⁐ゎᾮࢆ 13200 rpm, 10 min 㐲ᚰࡋࡓࠋ㐲ᚰᚋࠊ➼㔞ࡢ ୖΎ࡟10 unit ࡢ࢔ࣝ࢝ࣜࣇ࢛ࢫࣇ࢓ࢱ࣮ࢮࡲࡓࡣࠊ400 unit ࡢ Lambda phosphatase ࢆຍ࠼ࠊ 30Υࠊ2 ᫬㛫཯ᛂࡉࡏࡓࠋ཯ᛂࡣ 2× SDS sample buffer ࢆຍ࠼ࡿࡇ࡜࡛೵Ṇࡉࡏࠊࢧࣥࣉࣝࡢ ࣜࣥ㓟໬≧ែࡣ࢖࣒ࣀࣈࣟࢵࢺ࡟ࡼࡗ࡚ゎᯒࡋࡓࠋ

11

55. ࢖࣒ࣀࣈࣟࢵࢺゎᯒ

ࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࢆ⾜ࡗࡓ⣽⬊ࢆྛᅗ࡟♧ࡋࡓ⃰ᗘ࡜᫬㛫࡟ࡼࡾྛ✀㜼ᐖ๣ฎ⌮ᚋࠊ⣽⬊ ࢆ่⃭ࡋࡓࠋ⣽⬊ࢆị෭PBS ୍࡛ᅇὙίࡋࠊ1% (w/v) SDS / 20 mM Tris-HCl / 1 mM EDTA ⁐ ᾮ (pH 8.0) ࡛⁐ゎࡋࡓࠋ⣽⬊◚○ᾮࢆ෾⤖஝⇱ࡉࡏࠊ1×SDS sample buffer ࡟⁐ゎࡉࡏࡓࠋ⣽ ⬊◚○ᾮ୰ࡢ⥲⺮ⓑ㔞ࡣBCA protein assay kit (Thermo Scientific) ࢆ⏝࠸࡚ ᐃࡋࡓࠋⓎ⌧

ࣉࣛࢫ࣑ࢻࡢ⺮ⓑ㉁Ⓨ⌧ࢆ☜ㄆࡍࡿⅭࠊ➼㔞ࡢ⺮ⓑ㉁ࢆ 7.5% ࡶࡋࡃࡣ 10% SDS-࣏ࣜ࢔ࢡࣜ

ࣝ࢔࣑ࢻࢤࣝ㟁ẼὋື (SDS-PAGE) ࡛ศ㞳ࡋࡓࠋࣈࣟࢵࢺᚋࠊPVDF ࣓ࣥࣈࣞࣥࢆ 3%ࢫ࣒࢟ ࣑ࣝࢡࡶࡋࡃࡣ5% Bovine serum albumin (BSA) ࢆῧຍࡋࡓ TBS-T (Tris buffered saline ; 20 mM Tris-HCl, pH7.5, 0.15 M NaCl, 0.01% NP-40) ୰࡛ࣈࣟࢵࢡࡋࡓࠋMyc-ࢱࢢࢆ௜ຍࡋࡓ FLJ00018 ࡣ୍ḟᢠయ࡟ 1:2000 ᕼ㔘ࡋࡓ Anti-Myc ࣔࣀࢡࣟࢼ࣮ࣝᢠయ (Roche, 0.2 Pg/ml) ࢆ⏝࠸ࠊ஧ḟᢠయ࡟1:5000 ᕼ㔘ࡋࡓ horseradish peroxidase (HRP)-ᶆ㆑ࣔࣀࢡࣟࢼ࣮ࣝᢠ࣐

࢘ࢫIgG ᢠయ (MBL) ࢆ⏝࠸᳨࡚ฟࡋࡓࠋHalo-ࢱࢢࢆ௜ຍࡋࡓ FLJ00018 ࡶࡋࡃࡣ Vav3 ࡣ୍

ḟᢠయ࡟1:2000 ᕼ㔘ࡋࡓ Anti-Halo ࣏ࣜࢡࣟࢼ࣮ࣝᢠయ (Promega, 0.2 Pg/ml) ࢆ⏝࠸ࠊ஧ḟ

ᢠయ࡟1:5000 ᕼ㔘ࡋࡓ HRP-ᶆ㆑࣏ࣜࢡࣟࢼ࣮ࣝᢠ࢘ࢧࢠ IgG ᢠయ (MBL) ࢆ⏝࠸᳨࡚ฟࡋࡓࠋ

EGFR ࡣ୍ḟᢠయ࡟ 1:2000 ᕼ㔘ࡋࡓ Anti-EGFR ࣔࣀࢡࣟࢼ࣮ࣝᢠయ (Santa Cruz, 1000 Pg/ml) ࢆ⏝࠸ࠊ஧ḟᢠయ࡟ 1:5000 ᕼ㔘ࡋࡓ HRP-ᶆ㆑ࣔࣀࢡࣟࢼ࣮ࣝᢠ࣐࢘ࢫ IgG ࢆ⏝࠸᳨࡚

ฟࡋࡓࠋࣜࣥ㓟໬ࢳࣟࢩࣥ⺮ⓑ㉁ࡢ᳨ฟ࡟ࡣࠊ୍ḟᢠయ࡟1:2000 ᕼ㔘ࡋࡓ Anti-P-Tyr (PY20) ࣔ

ࣀࢡࣟࢼ࣮ࣝᢠయ (Santa Cruz Biotechnology, stock 100 Pg/ml) ࢆ⏝࠸ࠊ஧ḟᢠయ࡟ 1:5000 ᕼ

㔘ࡋࡓ HRP-ᶆ㆑ࣔࣀࢡࣟࢼ࣮ࣝᢠ࣐࢘ࢫ IgG ᢠయࢆ⏝࠸ࡓࠋྛ✀⺮ⓑ㉁ࡢྍど໬ࡣࠊ

enzyme-linked chemiluminescence ヨ ⸆ (GE Healthcare) ࢆ ⏝ ࠸ ࡚ LAS-4000 image analyzer (Fuji Film) ᳨࡛ฟࡋࡓࠋ

6. Mn2+ _{Phos-tag SDS-PAGE}

ࣜࣥ㓟໬ࡉࢀࡓ FLJ00018 ࡢ᭷↓ࢆ☜࠿ࡵࡿⅭࠊ᪂つ࡟㛤Ⓨࡉࢀࡓ࢖࣒ࣀࣈࣟࢵࢺᢏ⾡ࢆ⏝

12 ୰࡟ΰྜࡉࡏࠊ㟁ẼὋືࢆ⾜࠺᪉ἲ࡛࠶ࡿ (33)ࠋ㟁ẼὋື୰࡟ࣜࣥ㓟໬⺮ⓑ㉁ࡣ AAL107 ศᏊ ࡜┦஫స⏝ࡋࠊ㏻ᖖࡢ⺮ⓑ㉁ࡢ㟁ẼὋື࡜ẚ࡭⛣ືᗘࡀ㐜ࡃ࡞ࡿࡢ࡛ࠊࣜࣥ㓟໬⺮ⓑ㉁ࢆどぬⓗ ࡟᳨ฟࡍࡿࡇ࡜ࡀ࡛ࡁࡿࠋࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࢆ⾜ࡗࡓ⣽⬊࡟ྛᅗ࡟♧ࡋࡓ⃰ᗘ࡜᫬㛫࡟ࡼࡾ ྛ✀㜼ᐖ๣ฎ⌮ࠊ⣽⬊่⃭ࢆ⾜ࡗࡓࠋ⣽⬊ࢆị෭PBS ୍࡛ᅇὙίࡋࠊ1% (w/v) SDS H2O ⁐ᾮ ࡛⁐ゎࡋࡓࠋ⣽⬊⁐ゎᾮࢆ෾⤖஝⇱ࡉࡏࠊ1× SDS sample buffer ࡟⁐ゎࡉࡏࡓࠋ⣽⬊◚○ᾮ୰ ࡢ⥲⺮ⓑ㔞ࡣBCA protein assay kit (Thermo Scientific) ࢆ⏝࠸࡚ ᐃࡋࡓࠋ㔝⏕ᆺ FLJ00018 (FLJ00018 WT) ࣜࣥ㓟໬ࡢ᳨ฟ࡟ࡣࠊ࢔࣮࢞ࣟࢫࢆຍ࠼ࡓప⃰ᗘࡢ࢔ࢡࣜࣝ࢔࣑ࢻࢤࣝࢆ⏝࠸ ࡓ (34,35)ࠋ13 PM Phos-tag AAL107 (Wako), 26 PM MnCl2, 1.5% ࢔࣮࢞ࣟࢫࢆຍ࠼ࡓ 3% SDS-PAGE ࡛ศ㞳ࡋࡓࠋFLJ00018 ࡢྛ✀ኚ␗యࡢࣜࣥ㓟໬᳨ฟ࡟ࡣࠊ15 PM Phos-tag AAL107

(Wako), 25 PM MnCl2, 6% SDS-࣏ࣜ࢔ࢡࣜࣝ࢔࣑ࢻࢤࣝࢆ⏝࠸ࡓࠋ㟁ẼὋືᚋࠊ1 mM EDTA ⁐ᾮ࡛ࢤࣝࢆ10 ศ㛫ὙίࡋࠊMn2+_{ࢆ㝖࠸ࡓᚋ࡛࢖࣒ࣀࣈࣟࢵࢺࢆ⾜ࡗࡓࠋ} 7. ⺯ග⺮ⓑ㉁࡟ࡼࡿ⣽⬊ᙧែࡢほᐹ ࢝ࣂ࣮࢞ࣛࢫୖ୍࡛㐣ᛶ࡟ᙉไⓎ⌧ࡉࡏࡓ⣽⬊ࢆị෭PBS ୍࡛ᅇὙίᚋࠊ30 ศ㛫ࠊ4% ࣃࣛ ࣒࣍ࣝ࢔ࣝࢹࣄࢻᅛᐃࡉࡏࡓࠋࡑࡢᚋ࢝ࣂ࣮࢞ࣛࢫࢆ Perma Fluor ࡛ฎ⌮ࡉࡏࠊ ⺯ග࢖࣓࣮ ࢪࢆ⺯ග㢧ᚤ㙾࡟ࡼࡾほᐹࡋࡓ (BZ-9000; KEYENCE)ࠋྛ sample ࠿ࡽࠊ↓సⅭ࡟㑅ᢥࡋࡓྜ ィ 10 ᯛࡢ⺯ග⏬ീࢆྲྀᚓࡋࠊྜィ 50 ⣽⬊ࡢ⣽⬊࡟ࡘ࠸࡚⤫ィゎᯒࢆ⾜ࡗࡓࠋ⣽⬊ࡢィ ࡣ Image J ࢯࣇࢺ࢙࢘࢔ࢆ⏝࠸࡚⾜ࡗࡓࠋ 8. ⤫ィ࡜ศᯒ ࢹ࣮ࢱࡣᑡ࡞ࡃ࡜ࡶ 3 ᅇࡢᐇ㦂࠿ࡽᖹᆒ್±S.D.ࡢ್ࢆ♧ࡋࡓࠋࢢ࣮ࣝࣉ㛫ࡢ㐪࠸ࡢ⤫ィࡣ student-T-test ࡟ᚑ࠸ࠊゎᯒࢆ⾜ࡗࡓࠋ᭷ពᕪࡢ P ್ࡣ<0.05 ࡜ࡋࡓࠋ

13

➨

➨

3 ⠇ ⤖ᯝ

1. FLJ00018 ࡢE1࢔ࢻࣞࢼࣜࣥཷᐜయ่⃭ࢆ௓ࡋࡓࢩࢢࢼࣝ࡟ࡼࡿไᚚ 㐣ཤࠊFLJ00018 ࡣࠊm2 ࣒ࢫ࢝ࣜࣥᛶ࢔ࢭࢳࣝࢥࣜࣥཷᐜయ่⃭ཬࡧࠊGEJࢧࣈࣘࢽࢵࢺ࡟ ࡼࡗ࡚άᛶ໬ࡉࢀࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿ (23)ࠋFLJ00018 ࡀࠊ௚ࡢ GPCR ่⃭࡟ࡼࡗ࡚άᛶ

໬ࡉࢀࡿ࠿ࢆㄪ࡭ࡿⅭࠊE1AR ࡜ FLJ00018 ࢆ NIH3T3 ⣽⬊ෆ࡟୍㐣ᛶ࡟ඹⓎ⌧ࡉࡏࠊRho

GTPase ࡢάᛶ໬ࡢᣦᶆ࡜࡞ࡿ SRE άᛶ (36) ࢆ ᐃࡋࡓࠋ୍⯡ⓗ࡟E1AR ࡣ GDs ඹᙺᆺཷᐜ

య࡜⪃࠼ࡽࢀ࡚࠾ࡾࠊGDs ࢩࢢࢼࣝࡣୗὶࡢ࢚ࣇ࢙ࢡࢱ࣮࡟࢔ࢹࢽࣝ㓟ࢩࢡ࣮ࣛࢮࢆ᭷ࡋࠊE1AR

ࢩࢢࢼࣝ࡟࠾࠸࡚ࠊGEJࡢᐤ୚ࡣᑡ࡞࠸࡜⪃࠼ࡽࢀ࡚࠸ࡿࠋ

FLJ00018 ࡜E1AR ࢆඹⓎ⌧ࡉࡏࡓ⣽⬊࡟ࠊEAR ࢔ࢦࢽࢫࢺ࡛࠶ࡿ Isoproterenol (20 PM) ࡛ ่⃭ࡍࡿ࡜ࠊ⺮ⓑ㉁Ⓨ⌧࡟ኚ໬ࡣぢࡽࢀ࡞࠸࡟ࡶ࠿࠿ࢃࡽࡎ (Fig. 1-1B) ↓่⃭ࡢ⣽⬊࡟ẚ࡭ࠊ

Fig. 1--1 NIH3T3 ⣽⣽⬊࡟࠾ࡅࡿ FLJ00018 ࢆࢆ௓ࡋࡓ SRE ౫౫Ꮡⓗ㑇ఏᏊ㌿෗άᛶ࡬ࡢ Issoproterenol ࡜ E-ARKct ࡢᙳ㡪

A, NIH3T3 ⣽⬊࡟ pSRE.L-luciferase, pRL-SV40 ࣉࣛࢫ࣑ࢻ DNA, FLJ00018, E-ARKct, EAR ࡢⓎ⌧࣋ ࢡࢱ࣮ࢆࡑࢀࡒࢀ♧ࡋࡓࡼ࠺࡟ࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡋࡓࠋࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡋࡓ⣽⬊࡟ࡣࠊ⤊⃰ᗘ

20 PM, 5.5 ᫬㛫ࡢ Isoproterenol ่⃭ࢆ⾜ࡗࡓࠋࣝࢩࣇ࢙࣮ࣛࢮάᛶࡣ dual-luciferase reporter assay

system ࡟ࡼࡗ࡚ ᐃࡋࠊᚓࡽࢀࡓάᛶࡣ Mock ࢆ 1.0 ࡜ࡋ࡚ᶆ‽໬ࡋࡓ್ࢆ♧ࡋࡓࠋᐇ㦂ࡣᑡ࡞ࡃ࡜ࡶ 3 ᅇ⾜࠸ࠊ್ࢆᖹᆒ್± S.D.࡛♧ࡋࡓࠋ

B, NIH3T3 ⣽⬊࡟E1AR ࡑࡋ࡚ Myc ࢱࢢࢆ௜ຍࡋࡓ FLJ00018 ࡢⓎ⌧࣋ࢡࢱ࣮ࢆᅗ࡟♧ࡋࡓࡼ࠺࡟୍㐣ᛶ

࡟ࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡋࡓࠋࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡋࡓ⣽⬊࡟Isoproterenol (20 PM) ࡛ 15 ศ㛫่⃭ࢆ

⾜ࡗࡓࠋ➼㔞ࡢ⺮ⓑ㉁ࢆ7.5% SDS-PAGE ࡛ศ㞳ࡋࡓࠋ࢖࣒ࣀࣈࣟࢵࢺࡣࠊAnti-Myc ᢠయࢆ⏝࠸࡚⾜ࡗ

ࡓࠋ

14

SRE άᛶࡢ᭷ព࡞ୖ᪼ࡀぢࡽࢀࡓ (Fig. 1-1A)ࠋࡇࡢάᛶୖ᪼ࡣ C-terminal of E-adrenergic receptor kinase (E-ARKct) ࢆຍ࠼ࡿࡇ࡜࡟ࡼࡾᢚไࡉࢀࡓࠋE-ARKct ࡣࠊ่⃭࡟ࡼࡾ㐟㞳ࡉࢀ

ࡓ୕㔞యG ⺮ⓑ㉁ GEJ࡜┦஫స⏝ࡍࡿࡇ࡜࡟ࡼࡾࠊGEJࡢᶵ⬟ࢆᢚไࡍࡿ࡜⪃࠼ࡽࢀ࡚࠸ࡿࠋࡋ

࠿ࡋࠊE-ARKct ࡣࠊIsoproterenol ่⃭࡟క࠺ SRE άᛶୖ᪼ࢆ᏶඲࡟ࡣᢚไࡋ࡞࠿ࡗࡓࠋE-ARKct

࡟ࡼࡾᢚไࡉࢀ࡞࠿ࡗࡓSRE άᛶୖ᪼ࡣࠊGEJ௨እࡢᶵᵓࡀ㛵୚ࡋ࡚࠸ࡿ࡜⪃࠼ࡽࢀࡿࠋࡇࡢ

⤖ᯝ࠿ࡽࠊE1AR ࢩࢢࢼࣝࡣ GEJ㠀౫Ꮡⓗ࡞ᶵᵓ࡟ࡼࡿ FLJ00018 ࡢάᛶ໬࡟㛵୚ࡍࡿࡇ࡜ࡀ♧

၀ࡉࢀࡓࠋ

22. FLJ00018 ࡢE1AR ࢆ௓ࡍࡿ EGF ཷᐜయ transactivation ᶵᵓ࡟ࡼࡿάᛶ໬

୍᪉ࠊ㏆ᖺࠊGPCR ࢩࢢࢼࣝ࡟ࡼࡗ࡚ EGFR ࡀάᛶ໬ࡉࢀࡿ࡜࠸࠺ transactivation ᶵᵓࡀ ▱ࡽࢀ࡚࠾ࡾ (37-42)ࠊtransactivation ࢆᘬࡁ㉳ࡇࡍ GPCR ࡜ࡋ࡚ࠊE1AR ࡸE2AR ࡀ▱ࡽࢀ࡚ ࠸ࡿࠋGPCR ࢩࢢࢼࣝ࡟ࡼࡾࠊEGFR ࡀάᛶ໬ࡉࢀࡿࡢ࡛࠶ࢀࡤࠊEGFR ࡢ⮬ᕫࣜࣥ㓟໬ᶵᵓ

࡟ࡼࡾEGFR ࡢࢳࣟࢩࣥṧᇶࡀࣜࣥ㓟໬ࡉࢀࡿ࡜⪃࠼ࡽࢀࡿࠋࡇࡢࢩࢢࢼࣝఏ㐩⤒㊰࡟ࡘ࠸࡚

᳨ウࡍࡿⅭࠊNIH3T3 ⣽⬊࡟ EGFR ࡜E1AR ࠾ࡼࡧ FLJ00018 ࢆඹⓎ⌧ࡉࡏࠊIsoproterenol

่⃭ (20 PM) ᚋ࡟ࠊ⣽⬊◚○ᾮࢆ⏝࠸࡚࢖࣒ࣀࣈࣟࢵࢺゎᯒࢆ⾜ࡗࡓࠋ

15

C D

E F

G

Fig. 11-22 FLJ00018 ࡣࡣࠊE1AR ่่⃭ࢆ௓ࡋࡓ EEGF ཷᐜయ ttransactivation ࡟ࡼࡾࠊࣜࣥ㓟໬࡜άᛶ໬ࢆ ཷࡅࡿࠋ

A, NIH3T3 ⣽⬊࡟ EGFR, E1AR ࡑࡋ࡚ Myc ࢱࢢࢆ௜ຍࡋࡓ FLJ00018 ࡢⓎ⌧࣋ࢡࢱ࣮ࢆᅗ࡟♧ࡋࡓ

ࡼ࠺࡟୍㐣ᛶ࡟ࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡋࡓࠋࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡋࡓ⣽⬊࡟Isoproterenol (20 PM)

࡛0-30 ศ㛫่⃭ࢆ⾜ࡗࡓࠋ➼㔞ࡢ⺮ⓑ㉁ࢆ 7.5% SDS-PAGE ࡛ศ㞳ࡋࡓࠋ࢖࣒ࣀࣈࣟࢵࢺࡣࡑࢀࡒࢀࠊ

Anti-P-Tyr (PY20) ᢠయࡶࡋࡃࡣ Anti-EGFR ᢠయࢆ⏝࠸࡚⾜ࡗࡓࠋ

B, NIH3T3 ⣽⬊࡟ pSRE.L-luciferase, pRL-SV40 ࣉࣛࢫ࣑ࢻ DNA, FLJ00018, EAR, EGFR ࡢⓎ⌧࣋ ࢡࢱ࣮ࢆࡑࢀࡒࢀ♧ࡋࡓࡼ࠺࡟ࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡋࡓࠋࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡋࡓ⣽⬊࡟ࠊEGFR

࢟ࢼ࣮ࢮ㜼ᐖ๣ࡢAG1478 (10 PM, 1 ᫬㛫) ࢆ๓ฎ⌮ᚋࠊ⤊⃰ᗘ 20 PM, 5.5 ᫬㛫ࡢ Isoproterenol ่⃭

ࢆ⾜ࡗࡓࠋࣝࢩࣇ࢙࣮ࣛࢮάᛶࡣdual-luciferase reporter assay system ࡟ࡼࡗ࡚ ᐃࡋࠊᚓࡽࢀࡓά

ᛶࡣMock ࢆ 1.0 ࡜ࡋ࡚ᶆ‽໬ࡋࡓ್ࢆ♧ࡋࡓࠋᐇ㦂ࡣᑡ࡞ࡃ࡜ࡶ 3 ᅇ⾜࠸ࠊ್ࢆᖹᆒ್± S.D.࡛♧ࡋ

ࡓࠋ#, p<0.05ࠋ

C, E, G, NIH3T3 ⣽⬊ (C, G) ཬࡧ HEK293 ⣽⬊ (E) ࡟ EGFR, E1AR, GE1J2ࡑࡋ࡚Myc ࢱࢢࢆ௜ຍ

ࡋࡓ FLJ00018 ࡢⓎ⌧࣋ࢡࢱ࣮ࢆᅗ࡟♧ࡋࡓࡼ࠺࡟୍㐣ᛶ࡟ࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡋࡓࠋࢺࣛࣥࢫࣇ

࢙ࢡࢩࣙࣥࡋࡓ⣽⬊࡟10 PM, 30 ศ㛫ࡢ AG1478 ๓ฎ⌮ࢆ⾜ࡗࡓ (G)ࠋࡑࡢᚋ Isoproterenol (20 PM)

ࢆ⏝࠸࡚15 ศ㛫ࡢ่⃭ࢆ⾜ࡗࡓ (C, E, G)ࠋ➼㔞ࡢ⺮ⓑ㉁ࢆ 7.5% SDS-PAGE ࡛ศ㞳ࡋࡓࠋMyc ࢱࢢ

16

Isoproterenol ่⃭࡟ࡼࡾࠊEGFR ࡢࢳࣟࢩࣥࣜࣥ㓟໬ࡣࠊ᫬㛫౫Ꮡⓗ࡟ቑຍࡋࠊ15 ศ࡛ࣆ࣮

ࢡࢆ♧ࡋࡓࠋࡇࡢ⤖ᯝ࠿ࡽࠊE1AR ่⃭࡟ࡼࡾ EGFR ࡢ transactivation ᶵᵓࡀᘬࡁ㉳ࡇࡉࢀࡿ

ࡇ࡜ࡀ♧၀ࡉࢀࡓ (Fig. 1-2A)ࠋࡑࡇ࡛ࠊIsoproterenol ่⃭࡟ࡼࡿ FLJ00018 ࡢάᛶ໬࡟࠾࠸࡚ࠊ EGFR ࢩࢢࢼࣝࡢ㛵୚ࡀ࠶ࡿ࠿ࢆㄪ࡭ࡿⅭ࡟ࠊEGFR ࢟ࢼ࣮ࢮάᛶࡢ≉␗ⓗ㜼ᐖ๣࡛࠶ࡿ

AG1478 ࢆ⏝࠸ࠊSRE άᛶ࡬ࡢᙳ㡪ࢆ᳨ウࡋࡓࠋFLJ00018 ࡜E1AR ࠾ࡼࡧ EGFR ࢆ NIH3T3

⣽⬊࡟ඹⓎ⌧ࡉࡏ Isoproterenol (20 PM) ่⃭ࢆ⾜࠺࡜ࠊSRE άᛶࡢୖ᪼ࡀぢࡽࢀࡓࠋࡋ࠿ࡋ

࡞ࡀࡽࠊAG1478 (10 PM) ๓ฎ⌮ࢆ⾜ࡗࡓ⣽⬊࡛ࡣࠊIsoproterenol (10 PM) ่⃭࡟క࠺ SRE άᛶୖ᪼ࡢᢚไࡀぢࡽࢀࡓ (Fig. 1-2B)ࠋྠᵝࡢ᮲௳ࡢ⣽⬊࡟࠾ࡅࡿ FLJ00018 ⺮ⓑ㉁Ⓨ⌧㔞ࢆ ㄪ࡭ࡓ⤖ᯝࠊIsoproterenol ่⃭ࡋࡓ⣽⬊࡟࠾࠸࡚ࠊFLJ00018 ࡢ㟁ẼὋືୖࡢ⛣ືᗘࡢ㧗ศᏊ 㔞ഃ࡬ࡢࢩࣇࢺࡀぢࡽࢀࡓ (Fig. 1-2C)ࠋࡇࡢ⛣ືᗘࢩࣇࢺࡣ GEJ࡜ FLJ00018 ࢆඹⓎ⌧ࡋࡓ⣽ ⬊࡛ࡣぢࡽࢀ࡞࠿ࡗࡓࠋࡇࢀࡽࡢ⤖ᯝ࠿ࡽࠊEGFR transactivation ⤒㊰࡟ࡼࡗ࡚ FLJ00018 ࡀ ఱࡽ࠿ࡢಟ㣭ࢆཷࡅ࡚άᛶ໬ࡉࢀࡿࡇ࡜ࡀ♧၀ࡉࢀࡓࠋ㏆ᖺࠊVav ࡸ Asef, Tiam-1, E-Pix ࡞࡝

ࡢRhoGEF ࡀ⣽⬊እ่⃭࡟ࡼࡾࣜࣥ㓟໬ࡉࢀࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠾ࡾࠊRhoGEF ࡢࣜࣥ㓟໬ࡀ RhoGEF ࡢ ᶵ ⬟ ࡟ ࠾ ࠸ ࡚ 㔜 せ ࡛ ࠶ ࡿ ࡜ ⪃ ࠼ ࡽ ࢀ ࡿ (27,28,43) ࠋ FLJ00018 ࡀ ࠊ EGFR transactivation ࡟ࡼࡗ࡚ࣜࣥ㓟໬ࢆཷࡅࡿ࠿࡟ࡘ࠸᳨࡚ウࡍࡿⅭ࡟ࠊࢭࣜࣥࠊࢫࣞ࢜ࢽࣥṧᇶ ࡢ ࣜ ࣥ 㓟 ᇶࢆ ⬺ 㞳ࡉ ࡏࡿ ࢔ ࣝ ࢝ ࣜࣇ ࢛ ࢫࣇ ࢓ࢱ ࣮ ࢮ ཬࡧ Lambda ࣇ࢛ࢫࣇ࢓ࢱ࣮ࢮࢆ Isoproterenol ่⃭ࡋࡓ⣽⬊ࡢ⁐ゎᾮ࡟ฎ⌮ࡋࠊ཯ᛂᾮࢆ࢖࣒ࣀࣈࣟࢵࢺゎᯒࡍࡿࡇ࡜࡛ࠊヨᩱ Fig. 11-22 ⥆⥆ࡁ

D, NIH3T3 ⣽⬊࡟ EGFR, E1AR ࡑࡋ࡚ Myc ࢱࢢࢆ௜ຍࡋࡓ FLJ00018 ࡢⓎ⌧࣋ࢡࢱ࣮ࢆᅗ࡟♧ࡋࡓ

ࡼ࠺࡟୍㐣ᛶ࡟ࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡋࡓࠋࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡋࡓ⣽⬊࡟Isoproterenol (20 PM)

ࢆ⏝࠸࡚15 ศ㛫ࡢ่⃭ࢆ⾜ࡗࡓࠋ⣽⬊ࡢ⁐ゎᚋࠊ⁐ゎᾮ࡟ࠊ࢔ࣝ࢝ࣜࣇ࢛ࢫࣇ࢓ࢱ࣮ࢮ (10 unit) ࡶ

ࡋࡃࡣࠊLambda ࣇ࢛ࢫࣇ࢓ࢱ࣮ࢮ (400 unit) ࢆຍ࠼ࠊ30Υࠊ2 ᫬㛫཯ᛂࡉࡏࡓࠋ཯ᛂࡣࠊSDS ࢧࣥ ࣉࣝࣂࢵࣇ࢓࣮ࢆຍ࠼ࡿࡇ࡜࡛೵Ṇࡉࡏࠊࡑࢀࡒࢀࡢ⺮ⓑ㉁ࡢࣜࣥ㓟໬≧ែࢆ࢖࣒ࣀࣈࣟࢵࢺ࡟ࡼࡗ࡚ ☜ㄆࡋࡓࠋ

F, HEK293 ⣽⬊࡟ EGFR, E1AR ࡑࡋ࡚ Myc ࢱࢢࢆ௜ຍࡋࡓ FLJ00018 ࡢⓎ⌧࣋ࢡࢱ࣮ࢆᅗ࡟♧ࡋࡓ

ࡼ࠺࡟୍㐣ᛶ࡟ࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡋࡓࠋࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡋࡓ⣽⬊࡟Isoproterenol (20 PM)

ࢆ⏝࠸࡚15 ศ㛫ࡢ่⃭ࢆ⾜ࡗࡓࠋ➼㔞ࡢ⺮ⓑ㉁ࢆ 13 PM Phos-tagAAL107, 26 PM MnCl2, 1.5%࢔࢞

࣮ࣟࢫࢆຍ࠼ࡓ3% SDS-PAGE ࡛ศ㞳ࡋࡓࠋMyc ࢱࢢࡀ௜ຍࡉࢀࡓ FLJ00018 ࢆ᳨ฟࡍࡿⅭ࡟࢖࣒ࣀ

17

୰ࡢࣜࣥ㓟໬≧ែࡢ☜ㄆࢆ⾜ࡗࡓࠋࡑࡢ⤖ᯝࠊ࡝ࡕࡽࡢࣇ࢛ࢫࣇ࢓ࢱ࣮ࢮฎ⌮࡟࠾࠸࡚ࡶࠊ Isoproterenol ่⃭࡟ࡼࡿ㧗ศᏊ㔞ഃ࡬ࡢ⛣ືᗘࢩࣇࢺࡀぢࡽࢀ࡞ࡃ࡞ࡗࡓ (Fig. 1-2D)ࠋࡲࡓࠊ Phos-tag ࢤࣝࢆ⏝࠸ࡓ࢖࣒ࣀࣈࣟࢵࢺゎᯒࢆ⾜࠸ࠊࢧࣥࣉࣝ୰ࡢ⺮ⓑ㉁ࡢࣜࣥ㓟໬ࡢ᭷↓ࡢ☜

ㄆࡶ⾜ࡗࡓࠋPhos-tag ࢤࣝ࡟ࡣࠊMn2+_{࡜㘒యࢆᙧᡂࡉࡏࡓPhos-tag AAL107 ศᏊࡀྵࡲࢀ࡚}

࠸ࡿࠋ㟁ẼὋື୰࡟ࣜࣥ㓟໬⺮ⓑ㉁ࡣ AAL107 ศᏊ࡜┦஫స⏝ࡋࠊ㏻ᖖࡢ⺮ⓑ㉁ࡢ㟁ẼὋື࡜

ẚ࡭⛣ືᗘࡀ㐜ࡃ࡞ࡿࡢ࡛ࠊࣜࣥ㓟໬⺮ⓑ㉁ࢆどぬⓗ࡟᳨ฟࡍࡿࡇ࡜ࡀ࡛ࡁࡿ (33-35)ࠋ

Isoproterenol ่⃭ࡋࡓ HEK293 ⣽⬊ࡢ⁐ゎᾮࢆㄪ〇ࡋࠊPhos-tag ࢤࣝ࡟࠾࠸࡚ゎᯒࢆ⾜ࡗࡓ

࡜ࡇࢁࠊ㏻ᖖࡢ㟁ẼὋື᫬࡟ぢࡽࢀࡓ Isoproterenol ่⃭࡟క࠺ FLJ00018 ࡢ⛣ືᗘࢩࣇࢺࡀ

(Fig. 1-2E)ࠊࡼࡾ㢧ⴭ࡟ぢࡽࢀࠊFLJ00018 ࡢࣜࣥ㓟໬ࡀ♧၀ࡉࢀࡓ (Fig. 1-2F)ࠋFLJ00018

ࡀEGFR transactivation ࡟ࡼࡾάᛶ໬ࡉࢀ࡚࠸ࡿ࡜࠸࠺᭦࡞ࡿドᣐࢆᚓࡿⅭ࡟ࠊAG1478 ࢆ⏝

࠸࡚࢖࣒ࣀࣈࣟࢵࢺゎᯒࢆ⾜ࡗࡓࠋ Isoproterenol ่⃭࡟క࠺ FLJ00018 ࡢ⛣ືᗘࢩࣇࢺࡣࠊ AG1478 ๓ฎ⌮࡟ࡼࡗ࡚ᢚไࡉࢀࡓ (Fig. 1-2G)ࠋࡇࢀࡽࡢ⤖ᯝࡼࡾࠊFLJ00018 ࡀ EGFR transactivation ᶵᵓࢆ௓ࡋ࡚ࢭࣜࣥࠊࢫࣞ࢜ࢽࣥࣜࣥ㓟໬ࡉࢀࠊάᛶ໬ࡉࢀࡿࡇ࡜ࡀ♧၀ࡉࢀ ࡓࠋ

33. FLJ00018 ࡢ EGF ่⃭࡟ࡼࡿࣜࣥ㓟໬࡜άᛶ໬

Figure 1-2 ࡟࠾࠸࡚ࠊFLJ00018 ࡣ GPCR ࡟ࡼࡿ EGFR transactivation ࢆ௓ࡋ࡚άᛶ໬ࡉ

ࢀࡿࡇ࡜ࡀ♧၀ࡉࢀࡓࠋḟ࡟ࠊEGF ࡟ࡼࡿ EGFR ࡢ┤᥋่⃭࡟ࡼࡗ࡚ FLJ00018 ࡀάᛶ໬ࡉࢀ

ࡿ࠿ࢆ᳨ウࡍࡿⅭ࡟EGFR ࡜ FLJ00018 ࢆ୍㐣ᛶ࡟ඹⓎ⌧ࡉࡏࡓ NIH3T3 ⣽⬊ࢆ⏝࠸࡚ SRE

άᛶࢆ ᐃࡋࡓࠋ6 ᫬㛫ࡢ EGF (20 ng/ml) ่⃭࡟ࡼࡾࠊ㠀ᖖ࡟ᙉ࠸ SRE άᛶࡢୖ᪼ࡀぢࡽࢀ ࡓ (Fig. 1-3A)ࠋࡲࡓࠊEGF ࢩࢢࢼࣝ࡟ࡼࡗ࡚άᛶ໬ࡉࢀࡓ FLJ00018 ࡀࠊRhoGTPase ࡢ࡝ࡢ ࢧࣈࢱ࢖ࣉࢆάᛶ໬ࡍࡿ࠿ࢆㄪ࡭ࡿⅭ࡟ࠊRhoGTPase ࡢࢻ࣑ࢼࣥࢺࢿ࢞ࢸ࢕ࣈኚ␗యࢆ⏝࠸ࠊ SRE άᛶࢆ ᐃࡋࡓࠋࢻ࣑ࢼࣥࢺࢿ࢞ࢸ࢕ࣈኚ␗యࡣ G ⺮ⓑ㉁άᛶࢆḞ࠸ࡓࠊ୙άᛶ໬ᆺኚ␗

య࡛࠶ࡿࠋRhoA ࢻ࣑ࢼࣥࢺࢿ࢞ࢸ࢕ࣈኚ␗యࡣࠊEGFR ࢆ௓ࡋࡓ FLJ00018 ࡟ࡼࡿ SRE ౫Ꮡ

ⓗ㌿෗άᛶ࡟࠶ࡲࡾᙳ㡪ࢆཬࡰࡉ࡞࠿ࡗࡓࡢ࡟ᑐࡋࠊRac1 ࠾ࡼࡧ Cdc42 ࡢࢻ࣑ࢼࣥࢺࢿ࢞ࢸ࢕

18 ࠿ࡽࠊEGF ่⃭࡟ࡼࡗ࡚άᛶ໬ࡉࢀࡓ FLJ00018 ࡣ GEJ࡟ࡼࡿάᛶ໬࡜ྠᵝ࡟ࠊRac1, Cdc42 ࢆ≉␗ⓗ࡟άᛶ໬ࡉࡏࡿࡇ࡜ࡀ♧၀ࡉࢀࡓࠋ ḟ࡟ࠊEGF ่⃭᫬ࡢࠊFLJ00018 ࡢⓎ⌧ᙧែࢆ☜ㄆࡋࡓ࡜ࡇࢁࠊEGF (20 ng/ml) ่⃭࡟ࡼࡾ FLJ00018 ࡢ㟁ẼὋືୖࡢ⛣ືᗘࡀ㧗ศᏊ㔞ഃ࡟ኚ໬ࡋࡓ (Fig. 1-3C)ࠋࡲࡓࠊࡇࡢ⛣ືᗘኚ໬ ࡀࣜࣥ㓟໬࡟ࡼࡿࡶࡢ࠿࡝࠺࠿ࢆࠊ࢔ࣝ࢝ࣜࣇ࢛ࢫࣇ࢓ࢱ࣮ࢮཬࡧࠊLambda ࣇ࢛ࢫࣇ࢓ࢱ࣮ ࢮࢆ⏝࠸࡚☜ㄆࡋࡓࡇ࡜ࢁࠊ࢔ࣝ࢝ࣜࣇ࢛ࢫࣇ࢓ࢱ࣮ࢮཬࡧࠊLambda ࣇ࢛ࢫࣇ࢓ࢱ࣮ࢮࡢฎ ⌮࡟ࡼࡾࠊEGF ่⃭࡟ࡼࡿ FLJ00018 ࡢ㟁ẼὋືୖࡢ⛣ືᗘࢩࣇࢺࡀࡳࡽࢀ࡞ࡃ࡞ࡗࡓ (Fig. 1-3D)ࠋࡉࡽ࡟ࠊPhos-tag ࢤࣝࢆ⏝࠸࡚ HEK293 ⣽⬊࡟࠾ࡅࡿ EGF ่⃭ࢆ௓ࡋࡓ FLJ00018 ࡢ⛣ືᗘࢩࣇࢺࢆ☜ㄆࡋࡓ࡜ࡇࢁࠊ㏻ᖖࡢ㟁ẼὋື࡛ぢࡽࢀࡓ⛣ືᗘࢩࣇࢺ࡜ẚ㍑ࡋ࡚ࠊࡼࡾ㢧 ⴭ࡞ FLJ00018 ࡢ⛣ືᗘࢩࣇࢺࡀࡳࡽࢀࡓ (Fig. 1-3E, F)ࠋࡇࢀࡽࡢ⤖ᯝ࠿ࡽࠊFLJ00018 ࡀ EGF ࡟ࡼࡿ┤᥋่⃭࡟ࡼࡗ࡚ࡶࠊࣜࣥ㓟໬ࢆཷࡅ࡚άᛶ໬ࡉࢀࡿࡇ࡜ࡀ♧၀ࡉࢀࡓࠋࡑࡇ࡛ࠊ ⣽⬊ෆ࡟࠾ࡅࡿEGF ่⃭᫬ࡢ FLJ00018 ࣜࣥ㓟໬ࡢ᫬㛫ኚ໬࡟ࡘ࠸࡚ࠊFLJ00018 ࡜ EGFR ࢆඹⓎ⌧ࡉࡏࡓ⣽⬊࡟ࡉࡲࡊࡲ࡞᫬㛫࡟࠾࠸࡚EGF ่⃭ࢆຍ࠼ࡿࡇ࡜࡟ࡼࡾ᳨ウࢆ⾜ࡗࡓࠋࡑ ࡢ⤖ᯝࠊFLJ00018 ࡣ EGF ่⃭ᚋ 2 ศ࠿ࡽ 15 ศࡢ㛫࡛᫬㛫౫Ꮡⓗ࡟ࣜࣥ㓟໬ࡉࢀࠊ5 ศ࡛ࣆ ࣮ࢡ࡟㐩ࡋࡓ (Fig. 1-3G)ࠋE1AR ่⃭࡟ࡼࡿ EGFR ࡢάᛶ໬ (Fig. 1-2E) ࡟ẚ࡭ࠊEGF ࡢ┤᥋

่⃭࡟ࡼࡿ FLJ00018 ࡢࣜࣥ㓟໬ࡣ㠀ᖖ࡟᪩࠸཯ᛂ࡛࠶ࡾࠊࡇࡢᕪࡀ⣽⬊ᶵ⬟ࡢᕪ࡟࠿࠿ࢃࡗ

࡚࠸ࡿ࠿ࡶࡋࢀ࡞࠸ࡇ࡜ࢆ♧၀ࡋ࡚࠸ࡿࠋEGFR ࡣཷᐜయᆺࢳࣟࢩࣥ࢟ࢼ࣮ࢮ࡛࠶ࡿࡢ࡛ࠊ FLJ00018 ࡀ EGFR ࡟ࡼࡗ࡚┤᥋ࢳࣟࢩࣥࣜࣥ㓟໬ࡉࢀࡿ࠿ࢆ᳨ウࡋࡓࠋFigure 1-3H ࡟♧ࡋ ࡓࡼ࠺࡟ࠊVav3 ࡜ EGFR ࢆⓎ⌧ࡉࡏࡓ⣽⬊࡛ࡣࠊEGF ่⃭࡟ࡼࡾࠊࢳࣟࢩࣥࣜࣥ㓟໬ Vav3 ࡀ᳨ฟࡉࢀࡓࡢ࡟ᑐࡋࠊEGFRࠊFLJ00018 ࢆⓎ⌧ࡉࡏࡓ⣽⬊࡬ࡢ EGF ่⃭࡛ࡣࠊFLJ00018 ࡢࢳࣟࢩࣥࣜࣥ㓟໬ࡣ᳨ฟࡉࢀ࡞࠿ࡗࡓࠋࡇࢀࡽࡢ⤖ᯝࡣᑡ࡞ࡃ࡜ࡶࡇࡢᐇ㦂᮲௳ୗ࡟࠾࠸࡚ࡣࠊ EGFR ่⃭࡟ࡼࡾࠊFLJ00018 ࡣࢳࣟࢩࣥࣜࣥ㓟໬ࡼࡾࠊࡴࡋࢁࢭࣜࣥࠊࢫࣞ࢜ࢽࣥࣜࣥ㓟໬ ࢆཷࡅࠊάᛶ໬ࡉࢀࡿࡇ࡜ࢆ♧၀ࡋ࡚࠸ࡿࠋ

19

20

4. FLJ00018 ࡢ H-Ras ࢆ௓ࡍࡿάᛶ໬

ḟ࡟ࠊEGF ࡟ࡼࡿ FLJ00018 ࡢάᛶ໬࡟ࠊ࡝ࡢࡼ࠺࡞ࢩࢢࢼࣝศᏊࡢ㛵୚ࡀ࠶ࡿ࠿࡟ࡘ࠸࡚

᳨ウࢆ⾜ࡗࡓࠋ୍⯡ⓗ࡟ࠊEGFR ࡢୗὶ࡛ Ras-MAPK ࢝ࢫࢣ࣮ࢻࡀάᛶ໬ࡉࢀࡿࡇ࡜ࡀ▱ࡽࢀ

࡚࠸ࡿ (38)ࠋࡑࡇ࡛ࠊNIH3T3 ⣽⬊࡟ FLJ00018 ࡜ᜏᖖⓗάᛶ໬ᆺ H-Ras (H-Ras CA)ࠊࡶࡋ

H

Fig. 11-33 FFLJ00018 ࡣ EEGF ࢩࢢࢼࣝ࡟ࡼࡗ࡚άᛶ໬ࡉࢀࡿࠋ

A, B, NIH3T3 ⣽ ⬊ ࡟ pSRE.L-luciferase, pRL-SV40 ࣉ ࣛ ࢫ ࣑ ࢻ DNA, FLJ00018, EGFR, pF4A-RhoA T19N, pF4A-Rac T17N, pF4A-Cdc42 T17N ࡢⓎ⌧࣋ࢡࢱ࣮ࢆࡑࢀࡒࢀ♧ࡋࡓࡼ࠺࡟ࢺ

ࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡋࡓࠋࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡋࡓ⣽⬊࡟ࠊ20 ng/ml, 6 ᫬㛫ࡢ EGF ่⃭ࢆ⾜ࡗࡓࠋ

ࣝࢩࣇ࢙࣮ࣛࢮάᛶࡣdual-luciferase reporter assay system ࢆ⏝࠸࡚ ᐃࡋࠊᚓࡽࢀࡓάᛶࡣ Mock

ࢆ1.0 ࡜ࡋ࡚ᶆ‽໬ࡋࡓ್ࢆ♧ࡋࡓࠋᐇ㦂ࡣᑡ࡞ࡃ࡜ࡶ 3 ᅇ⾜࠸ࠊ್ࢆᖹᆒ್± S.D.࡛♧ࡋࡓࠋ

C, E, G, H, NIH3T3 ⣽⬊ (C, G, H) ཬࡧ HEK293 ⣽⬊ (E) ࡟ EGFR, GE1J2ࡑࡋ࡚Myc ࢱࢢࡶࡋࡃ

ࡣHalo ࢱࢢࢆ௜ຍࡋࡓ FLJ00018, Halo ࢱࢢࡀ௜ຍࡉࢀࡓ Vav3 ࡢⓎ⌧࣋ࢡࢱ࣮ࢆᅗ࡟♧ࡋࡓࡼ࠺࡟

୍㐣ᛶ࡟ࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡋࡓࠋࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࢆ⾜ࡗࡓ⣽⬊࡟EGF (20 ng/ml) ࢆ⏝࠸

࡚ 15 ศ㛫 (C, E, H) ࡶࡋࡃࡣࠊᅗ࡟♧ࡋࡓ᫬㛫 (G) ่࡛⃭ࢆ⾜ࡗࡓࠋ➼㔞ࡢ⺮ⓑ㉁ࡣ 7.5%

SDS-PAGE ࡛ศ㞳ࡋࡓࠋMyc ࢱࢢࡀ௜ຍࡉࢀࡓ FLJ00018 ࡢ᳨ฟ࡟ࡣ Anti-Myc ᢠయࢆࠊHalo ࢱࢢ

ࡀ௜ຍࡉࢀࡓ⺮ⓑ㉁ࡢ᳨ฟ࡟ࡣ Anti-Halo ᢠయࢆࠊࣜࣥ㓟໬ࢳࣟࢩࣥࢆྵࡴ⺮ⓑ㉁ࡢ᳨ฟ࡟ࡣ Anti-P-Tyr (PY20) ᢠయࢆ⏝࠸ࡓࠋ D, NIH3T3 ⣽⬊࡟ࠊEGFRࠊMyc ࢱࢢࢆ௜ຍࡋࡓ FLJ00018 ࡢⓎ⌧࣋ࢡࢱ࣮ࢆᅗ࡟♧ࡋࡓࡼ࠺࡟୍ 㐣ᛶ࡟ࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡋࡓࠋࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࢆ⾜ࡗࡓ⣽⬊࡟20 ng/ml ࡢ EGF ่⃭ (15 ศ㛫) ࢆ⾜ࡗࡓࠋ⣽⬊ࡢ⁐ゎᚋࠊ⁐ゎᾮ࡟ࠊ࢔ࣝ࢝ࣜࣇ࢛ࢫࣇ࢓ࢱ࣮ࢮ (10 unit) ࡶࡋࡃࡣࠊLambda ࣇ࢛ࢫࣇ࢓ࢱ࣮ࢮ (400 unit) ࢆຍ࠼ࠊ30Υࠊ2 ᫬㛫཯ᛂࡉࡏࡓࠋ཯ᛂࡣࠊSDS ࢧࣥࣉࣝࣂࢵࣇ࢓࣮ ࢆຍ࠼ࡿࡇ࡜࡛೵Ṇࡉࡏࠊࡑࢀࡒࢀࡢ⺮ⓑ㉁ࡢࣜࣥ㓟໬≧ែࢆ࢖࣒ࣀࣈࣟࢵࢺ࡟ࡼࡗ࡚☜ㄆࡋࡓࠋ F, HEK293 ⣽⬊࡟ EGFRࠊMyc ࢱࢢࢆ௜ຍࡋࡓ FLJ00018 ࡢⓎ⌧࣋ࢡࢱ࣮ࢆᅗ࡟♧ࡋࡓࡼ࠺࡟୍㐣 ᛶ࡟ࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡋࡓࠋࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࢆ⾜ࡗࡓ⣽⬊࡟20 ng/ml ࡢ EGF ่⃭ (15 ศ 㛫) ࢆ⾜ࡗࡓࠋ➼㔞ࡢ⺮ⓑ㉁ࢆ 13 PM Phos-tag AAL107, 26 PM MnCl2, 1.5%࢔࣮࢞ࣟࢫࢆຍ࠼ࡓ 3% SDS-PAGE ࡛ศ㞳ࡋࡓࠋMyc ࢱࢢࡀ௜ຍࡉࢀࡓ FLJ00018 ࢆ᳨ฟࡍࡿⅭ࡟࢖࣒ࣀࣈࣟࢵࢺࡣ Anti-Myc ᢠయࢆ⏝࠸࡚⾜ࡗࡓࠋ

21

ࡃࡣH-Ras ࢻ࣑ࢼࣥࢺࢿ࢞ࢸ࢕ࣈኚ␗య (H-Ras DN) ࢆඹⓎ⌧ࡉࡏࠊ࢖࣒ࣀࣈࣟࢵࢺゎᯒࢆ⾜

ࡗࡓࠋH-Ras CA ࡜ࡢඹⓎ⌧࡟ࡼࡾࠊFLJ00018 ࡢ⛣ືᗘࢩࣇࢺࡀぢࡽࢀࡓ (Fig. 1-4A)ࠋࡲࡓࠊ

H-Ras DN ࡜ࡢඹⓎ⌧࡟ࡼࡾ EGF ่⃭ (20 ng/ml) ࡟ࡼࡿ FLJ00018 ࡢ⛣ືᗘࢩࣇࢺࡢᢚไࡀ ぢࡽࢀࡓ (Fig. 1-4B)ࠋࡇࢀࡽࡢ⤖ᯝ࠿ࡽ H-Ras ࡀ EGF ่⃭࡟క࠺ FLJ00018 ࡢάᛶ໬࡟㛵୚ ࡋ࡚࠸ࡿࡇ࡜ࡀ♧၀ࡉࢀࡓࠋ

5. FLJ00018 ࡢ H-Ras/MAPK ⤒㊰ࢆ௓ࡍࡿάᛶ໬

EGFR-H-Ras ࡢୗὶ࡛ࡣࠊMAPK ࢝ࢫࢣ࣮ࢻࡀാࡃࡇ࡜ࡀ⪃࠼ࡽࢀࡿࡇ࡜࠿ࡽࠊMAPK ࢝ࢫ

ࢣ࣮ࢻࡢ୰ࡢMEK ࡟ᑐࡍࡿ㜼ᐖ๣࡛࠶ࡿ U0126 (10 PM) ࢆ 30 ศ㛫๓ฎ⌮ᚋࠊEGF ่⃭ࢆ⾜

࠸ (20 ng/ml, 15 ศ㛫)ࠊ࢖࣒ࣀࣈࣟࢵࢺἲࢆ⏝࠸࡚ FLJ00018 ࡢ㟁ẼὋືୖࡢ⛣ືᗘࢩࣇࢺࢆ

᳨ウࡋࡓࠋᑐ↷࡜ࡋ࡚⏝࠸ࡓࠊ࢝ࣝࣔࢪࣗࣜࣥ࢟ࢼ࣮ࢮϩ (CaMKϩ) ࡢ㜼ᐖ๣ KN-93 (10 PM)

ฎ⌮⣽⬊࡛ࡣࠊEGF ่⃭࡟క࠸ࠊFLJ00018 ࡢ⛣ືᗘࢩࣇࢺࡀぢࡽࢀࡓࡢ࡟ᑐࡋࠊU0126 (10 PM)

ฎ⌮⣽⬊࡟࠾࠸࡚ࡣࠊEGF ่⃭࡟ࡼࡿ⛣ືᗘࢩࣇࢺࡢᢚไࡀぢࡽࢀࡓ (Fig. 1-5A)ࠋࡇࡢ⤖ᯝ࠿

ࡽFLJ00018 ࡀ MEK ࡶࡋࡃࡣࡑࡢୗὶࡢࣜࣥ㓟໬㓝⣲࡟ࡼࡗ࡚ࣜࣥ㓟໬ࡉࢀࡿࡇ࡜ࡀ♧၀ࡉ

ࢀࡓࠋࡉࡽ࡟ࠊU0126 (10 PM, 1 ᫬㛫) ࡶࡋࡃࡣࠊKN-93 (10 PM, 1 ᫬㛫) ฎ⌮ࢆ⾜ࡗࡓ⣽⬊ࢆ

⏝࠸࡚ࠊSRE άᛶ ᐃࢆ⾜ࡗࡓ࡜ࡇࢁࠊ࡝ࡕࡽࡢ㜼ᐖ๣ࢆ⏝࠸ࡓሙྜ࡟ࡶ EGF ่⃭࡟క࠺ SRE

Fiig. 1-44 FFLJ00018 ࡣ HH-RRas ࢆ௓ࡋ࡚άᛶ໬ࡉࢀࡿࠋ

A, B, NIH3T3 ⣽⬊࡟ EGFR, ᜏᖖⓗάᛶ໬ᆺ H-Ras (H-Ras CA), ࢻ࣑ࢼࣥࢺࢿ࢞ࢸ࢕ࣈᆺ H-Ras (H-Ras DN), ࡑࡋ࡚ Myc ࢱࢢࢆ௜ຍࡋࡓ FLJ00018 ࡢⓎ⌧࣋ࢡࢱ࣮ࢆᅗ࡟♧ࡋࡓࡼ࠺࡟୍㐣ᛶ࡟ࢺ

ࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡋࡓࠋࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࢆ⾜ࡗࡓ⣽⬊࡟20 ng/ml, 15 ศ㛫ࡢ EGF ่⃭ࢆ⾜

ࡗࡓࠋ➼㔞ࡢ⺮ⓑ㉁ࡣ7.5% SDS-PAGE ࡛ศ㞳ࡋࡓࠋ Myc ࢱࢢࡀ௜ຍࡉࢀࡓ FLJ00018 ࡢ᳨ฟࡢⅭ

࡟ࠊAnti-Myc ᢠయࢆ⏝࠸ࡓࠋ

22 άᛶࡢୖ᪼ࡢᢚไࡀぢࡽࢀࡓ (Fig. 1-5B, C)ࠋࡇࢀࡽࡢ⤖ᯝࡣࠊKN-93 ๓ฎ⌮ࢆ⾜ࡗࡓ⣽⬊࡛ EGF ่⃭࡟ࡼࡿ FLJ00018 ࡢ⛣ືᗘࢩࣇࢺࡢᢚไࡀぢࡽࢀ࡞࠿ࡗࡓ⤖ᯝ࡜୍⮴ࡋ࡞࠸ࠋࡇࢀࡣࠊ MEK ࡶࡋࡃࡣࡑࡢୗὶࡢࣜࣥ㓟໬㓝⣲ࡣ FLJ00018 ࡢࣜࣥ㓟໬࡜ SRE άᛶࡢ୧᪉࡟ᙳ㡪ࢆ୚ ࠼ࡿࡢ࡟ᑐࡋࠊCaMKϩࡣࠊMEK ࡀ FLJ00018 ࢆࣜࣥ㓟໬ࡍࡿ⤒㊰࡜ࡣูࡢ⤒㊰ࢆ௓ࡋࠊ FLJ00018 ࣜࣥ㓟໬࡟ࡣᙳ㡪ࢆࡏࡎࠊSRE ࡟ࡢࡳᙳ㡪ࢆཬࡰࡋ࡚࠸ࡿࡇ࡜ࡀ⪃࠼ࡽࢀࡓࠋCaMK ϩࡀࠊEGF ่⃭࡟ࡼࡿ FLJ00018 ࢆ௓ࡋࡓ SRE άᛶࡢᢚไ࡟㛵୚ࡍࡿࡇ࡜࠿ࡽࠊEGF ࡢୗὶ

࡟࠾࠸࡚ࠊ௚ࡢMAPK ࡀാ࠸࡚࠸ࡿ࠿ࢆ᳨ウࡋࡓࠋFLJ00018 ࡜ EGFR ࢆඹⓎ⌧ࡉࡏࡓ⣽⬊࡟ࠊ p38-MAPK 㜼ᐖ๣ࠊSB202190 ཬࡧࠊJNK 㜼ᐖ๣ࠊSP600125 ࢆ 30 ศ๓ฎ⌮ࡋࡓᚋࠊEGF ่ ⃭ࢆ⾜࠸ࠊFLJ00018 ࡢ㟁ẼὋືୖࡢ⛣ືᗘࢩࣇࢺࢆ☜ㄆࡋࡓࠋࡑࡢ⤖ᯝࠊ࡝ࡕࡽࡢ㜼ᐖ๣ࡶࠊ EGF ่⃭࡟ࡼࡿ FLJ00018 ࡢ⛣ືᗘࢩࣇࢺ࡟ᙳ㡪ࡣぢࡽࢀ࡞࠿ࡗࡓ (Fig. 1-5D)ࠋࡲࡓྠᵝ࡟ࠊ FLJ00018 ࢆ௓ࡋࡓ SRE ౫Ꮡⓗ㑇ఏᏊ㌿෗άᛶ࡬ࡢ p38-MAPK ཬࡧ JNK ࡢᙳ㡪࡟ࡘ࠸࡚ࡶ᳨ ウࢆ⾜ࡗࡓࠋࡋ࠿ࡋ࡞ࡀࡽࠊ࡝ࡕࡽࡢ࢟ࢼ࣮ࢮ㜼ᐖ๣ࡶࠊSRE άᛶ࡟ᑐࡍࡿᙳ㡪ࡣぢࡽࢀࡎ (Fig. 1-5E)ࠊࡇࢀࡽ 2 ࡘࡢ࢟ࢼ࣮ࢮ⤒㊰ࡣ FLJ00018 ࡢࣜࣥ㓟໬ཬࡧάᛶ໬࡟ᙳ㡪ࢆ୚࠼࡞࠸ࡇ࡜ࡀ ♧၀ࡉࢀࡓࠋ A B _C

23 6. EGF ่⃭ࢆ௓ࡋࡓ FLJ00018 ࣜࣥ㓟໬࡟㛵ࢃࡿᵓ㐀ࡢྠᐃ ḟ࡟ࠊEGF ่⃭࡟ࡼࡿ FLJ00018 ࡢࣜࣥ㓟໬࡟㛵ࢃࡿᵓ㐀ࢆㄪ࡭ࡿⅭ࡟ࠊFLJ00018 ࡢྛ✀ Ḟᦆኚ␗యࢆEGFR ࡜ඹⓎ⌧ࡉࡏࡓ⣽⬊ࢆ⏝࠸࡚࢖࣒ࣀࣈࣟࢵࢺゎᯒ࡟ࡼࡾ FLJ00018 ࡢ⛣ື Fiig. 1--5 FLJ00018 ࡣࡣMAPK ⤒⤒㊰ࢆ௓ࡋ࡚άᛶ໬ࡉࢀࡿࠋ

A, D, NIH3T3 ⣽⬊࡟ EGFR, Myc ࢱࢢࢆ௜ຍࡋࡓ FLJ00018 ࡢⓎ⌧࣋ࢡࢱ࣮ࢆᅗ࡟♧ࡋࡓࡼ࠺࡟୍

㐣ᛶ࡟ࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡋࡓࠋࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥᚋࠊ⣽⬊࡟CaMKϩ㜼ᐖ๣ࠊKN-93 (10 PM,

A)ࠊMEK 㜼ᐖ๣ࠊU0126 (10 PM, A) ࡶࡋࡃࡣࠊp38MAPK 㜼ᐖ๣ࠊSB202190 (10 PM, D)ࠊJNK 㜼ᐖ๣ࠊSP600125 (10 PM, D) ࢆ 30 ศ㛫ฎ⌮ᚋࠊ⤊⃰ᗘ 20 ng/ml, 15 ศ㛫ࡢ EGF ่⃭ࢆ⾜ࡗࡓࠋ

➼㔞ࡢ⺮ⓑ㉁ࢆ 7.5% SDS-PAGE ࡛ศ㞳ࡋࡓࠋMyc ࢱࢢࡀ௜ຍࡉࢀࡓ FLJ00018 ࡢ᳨ฟࡢⅭ࡟ࠊ

Anti-Myc ᢠయࢆ⏝࠸ࡓࠋ

B, C, E, NIH3T3 ⣽⬊࡟ pSRE.L-luciferase, pRL-SV40 ࣉࣛࢫ࣑ࢻ DNA, FLJ00018, EGFR ࡢⓎ⌧ ࣋ࢡࢱ࣮ࢆࡑࢀࡒࢀ♧ࡋࡓࡼ࠺࡟ࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡋࡓࠋࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡋࡓ⣽⬊࡟ࠊ10 PM ࡢ KN-93 (B)ࠊ10 PM ࡢ U0126 (C)ࡶࡋࡃࡣࠊ10 PM ࡢ SB202190 (E)ࠊ10 PM ࡢ SP600125 (E)

ࢆ 1 ᫬㛫๓ฎ⌮ᚋࠊ⤊⃰ᗘ 20 ng/ml ࡢ EGF ่⃭ࢆ 6 ᫬㛫⾜ࡗࡓࠋࣝࢩࣇ࢙࣮ࣛࢮάᛶࡣ

dual-luciferase reporter assay system ࡟ࡼࡗ࡚ ᐃࡋࠊᚓࡽࢀࡓάᛶࡣ Mock ࢆ 1.0 ࡜ࡋ࡚ᶆ‽໬

ࡋࡓ್ࢆ♧ࡋࡓࠋᐇ㦂ࡣᑡ࡞ࡃ࡜ࡶ3 ᅇ⾜࠸ࠊ್ࢆᖹᆒ್± S.D.࡛♧ࡋࡓࠋ

D

E D

24

ᗘࢩࣇࢺࢆ᳨ウࡋࡓࠋFigure 1-6A ࡟ FLJ00018 ࡢྛኚ␗యࡢᵓ㐀ࢆ♧ࡋࡓࠋFigure 1-6B ࡟♧ ࡋࡓࡼ࠺࡟ࠊFLJ00018 WT ࡜ FLJ00018 P1 ኚ␗యࢆ EGFR ࡜ඹⓎ⌧ࡉࡏࡓ⣽⬊࡛ࡣ EGF ่ ⃭క࠸ࠊ⛣ືᗘࢩࣇࢺࡀぢࡽࢀࡓࠋ୍᪉࡛ࠊFLJ00018 P1.5, FLJ00018 P2, FLJ00018 P3, FLJ00018 'N ࢆ EGFR ࡜Ⓨ⌧ࡉࡏࡓ⣽⬊࡟࠾࠸࡚ࡣ่⃭࡟క࠺᫂☜࡞⛣ືᗘࢩࣇࢺࡣ࡯࡜ࢇ ࡝ぢࡽࢀ࡞࠿ࡗࡓࠋࡇࡢ⤖ᯝ࠿ࡽEGF ่⃭࡟ࡼࡿ FLJ00018 ࡢࣜࣥ㓟໬࡟㛵ࢃࡿ࢔࣑ࣀ㓟ࡢᑡ ࡞ࡃ࡜ࡶ1 ࡘࡣࠊFLJ00018 ࡢ 616-970 ␒┠ࡢ࢔࣑ࣀ㓟㓄ิෆ࡟Ꮡᅾࡍࡿྍ⬟ᛶࡀ♧၀ࡉࢀࡓࠋ A B C

25 ࡑࡇ࡛ࡉࡽ࡟ࠊP1.5, P2, P3, 'N ࡢ㓄ิ୰࡟ EGF ่⃭࡟ࡼࡿࣜࣥ㓟໬࡟㛵ࢃࡿᵓ㐀ࡀᏑᅾࡍ ࡿ࠿࡝࠺࠿ࢆPhos-tag ࢤࣝࢆ⏝࠸࡚☜ㄆࡋࡓࠋࡑࡢ⤖ᯝࠊPhos-tag ࢤࣝ୰࡟࠾࠸࡚ࠊP1.5, 'N ࢆྵࡴ⣽⬊࡬ࡢEGF ่⃭࡟ࡼࡾࠊ⛣ືᗘࢩࣇࢺࡀ☜ㄆࡉࢀࡓ (Fig. 1-6C)ࠋࡇࢀࡽࡢ⤖ᯝ࠿ࡽࠊ FLJ00018 ࡢ 616-970 ␒┠ࡢ࢔࣑ࣀ㓟㓄ิ௨እ࡟ 465-615, 965-1386 ␒┠ࡢ࢔࣑ࣀ㓟㓄ิ࡟ࡶࠊ EGF ่⃭࡟ࡼࡗ࡚ࣜࣥ㓟໬ࡉࢀࡿ㡿ᇦࡀᏑᅾࡍࡿྍ⬟ᛶࡀ♧၀ࡉࢀࡓࠋࡋ࠿ࡋ࡞ࡀࡽࠊP2, P3 ࢆẚ㍑ࡍࡿ࡜ࠊP2 ࡛ࡣ⛣ືᗘࡢ␗࡞ࡿ」ᩘࡢࢩࢢࢼࣝࡀ᳨ฟࡉࢀࠊࡇࡢࡼ࠺࡞ኚ໬ࡣ P3 ࡛ࡣ ぢࡽࢀ࡞࠿ࡗࡓࠋࡇࡢࡇ࡜࠿ࡽࠊ311-464 ␒┠ࡢ࢔࣑ࣀ㓟㓄ิ࡟ࡶࣜࣥ㓟໬ࡉࢀࡿᵓ㐀ࡀྵࡲࢀ ࡿྍ⬟ᛶࡀྰᐃ࡛ࡁࡎࠊ௒ᚋࡉࡽ࡞ࡿ᳨ウࡀᚲせ࡛࠶ࡿ࡜⪃࠼ࡽࢀࡿࠋ 77. EGF ่⃭࡟ࡼࡿ FLJ00018 ࣜࣥ㓟໬࡟㛵ࢃࡿ࢔࣑ࣀ㓟ࡢྠᐃ

Figure 1-5 ࡟࠾࠸࡚ࠊFLJ00018 ࡣ EGF ่⃭࡟ࡼࡾࠊMEK ࡢୗὶࡢศᏊ࡟ࡼࡗ࡚ࣜࣥ㓟໬ ࢆཷࡅࡿࡇ࡜ࡀ♧၀ࡉࢀࠊFigure 1-6 ࡟࠾࠸࡚ࠊࡑࡢࣜࣥ㓟໬࡟࠿࠿ࢃࡿ㒊఩ࡢᑡ࡞ࡃ࡜ࡶ୍ ࡘࡣࠊFLJ00018 ࡢ 615-970 ␒┠ࡢ࠸ࡎࢀ࠿ࡀ࠿࠿ࢃࡿྍ⬟ᛶࡀ♧၀ࡉࢀࡓࠋࡑࡇ࡛ࠊࡇࡢ㡿 ᇦ࡟࠾࠸࡚ࠊMEK ࡢᶆⓗศᏊࡢ୍ࡘ࡛࠶ࡿࠊERK ࡟ࡼࡿࣜࣥ㓟໬ࡢࢥࣥࢭࣥࢧࢫ㓄ิ (44) ࢆ ㄪ࡭ࡓ࡜ࡇࢁࠊFLJ00018 ࡢ 680 ␒┠ࡢࢫࣞ࢜ࢽࣥࡀ᥎ᐃࡉࢀࡓࠋࡑࡇ࡛ࠊࡇࡢ 680 ␒┠ࡢࢫ ࣞ࢜ࢽࣥࢆ࢔ࣛࢽࣥ࡟⨨᥮ࡋࡓⅬኚ␗య (FLJ00018 T680A) ࢆస〇ࡋࠊEGF ่⃭࡟ࡼࡿ㟁Ẽ Ὃືୖࡢ⛣ືᗘࢩࣇࢺࢆ࢖࣒ࣀࣈࣟࢵࢺゎᯒ࡟ࡼࡾ᳨ウࡋࡓ (Fig. 1-7A)ࠋࡑࡢ⤖ᯝࠊFLJ00018 T680A ኚ␗యࡣࠊFLJ00018 WT ࡜ẚ࡭ࠊEGF ่⃭࡟ࡼࡿ⛣ືᗘࢩࣇࢺࡢᢚไࡀࡳࡽࢀࡓࠋ Fiig. 1--6 EGF ่่⃭࡟ࡼࡿFLJ00018 ࣜࣜࣥ㓟໬࡟㛵ࢃࡿᵓ㐀ࡢྠᐃ A, FLJ00018 ࡢྛ✀ኚ␗యࡢᵓ㐀ࠋWT, P1, P1.5, P2, P3, 'N ࢥࣥࢫࢺࣛࢡࢺࡣࠊࡑࢀࡒࢀ FLJ00018 ࡢ1-1386, 1-970, 1-615, 1-464, 1-310, 965-1386 ࢔࣑ࣀ㓟ṧᇶࡀྵࡲࢀࡿࠋ

BNIH3T3 ⣽⬊࡟ EGFR, Myc ࢱࢢࢆ௜ຍࡋࡓ FLJ00018 ྛ✀ኚ␗యࡢⓎ⌧࣋ࢡࢱ࣮ࢆᅗ࡟♧ࡋࡓࡼ

࠺࡟୍㐣ᛶ࡟ࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡋࡓࠋࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡋࡓ⣽⬊࡟EGF (20 ng/ml) ࢆ⏝࠸

࡚ 15 ศ㛫่⃭ࢆ⾜ࡗࡓࠋ➼㔞ࡢ⺮ⓑ㉁ࢆ 7.5% SDS-PAGE ࡛ศ㞳ࡋࡓࠋMyc ࢱࢢࡀ௜ຍࡉࢀࡓ

FLJ00018 ࡢ᳨ฟࡢⅭ࡟ࠊAnti-Myc ᢠయࢆ⏝࠸ࡓࠋڸ, ⛣ືᗘኚ໬ࡢぢࡽࢀࡓࣂࣥࢻࠋڹ, ่⃭๓ࡢ FLJ00018 ࡢࣂࣥࢻࠋ

C, NIH3T3 ⣽⬊࡟ EGFR, Myc ࢱࢢࢆ௜ຍࡋࡓ FLJ00018 ࡢྛ✀ኚ␗యࡢⓎ⌧࣋ࢡࢱ࣮ࢆᅗ࡟♧ࡋࡓ

ࡼ࠺࡟୍㐣ᛶ࡟ࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡋࡓࠋࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࢆ⾜ࡗࡓ⣽⬊࡟20 ng/ml ࡢ EGF

่⃭ (15 ศ㛫) ࢆ⾜ࡗࡓࠋ➼㔞ࡢ⺮ⓑ㉁ࢆ 15 PM Phos-tag AAL107, 30 PM MnCl2 ࢆຍ࠼ࡓ 6%

SDS-PAGE ࡛ศ㞳ࡋࡓࠋMyc ࢱࢢࡀ௜ຍࡉࢀࡓ FLJ00018 ࢆ᳨ฟࡍࡿⅭ࡟࢖࣒ࣀࣈࣟࢵࢺࡣ Anti-Myc ᢠయࢆ⏝࠸࡚⾜ࡗࡓࠋڹ, ่⃭๓ࡢ FLJ00018 ࡢࣂࣥࢻࠋڸ, ่⃭࡟ࡼࡾ⛣ືᗘኚ໬ࡢぢࡽ

26

FLJ00018 ࡢ 680 ␒┠ࡢࢫࣞ࢜ࢽࣥṧᇶ࡟࠾࠸࡚ࠊFLJ00018 ࡀࣜࣥ㓟໬ࡉࢀࡿࡇ࡜ࢆࡉࡽ࡟ ☜࠿ࡵࡿⅭࠊHEK293 ⣽⬊࡟ EGFR ཬࡧࠊFLJ00018 WT ࡲࡓࡣ FLJ00018 T680A ࢆᑟධࡋ (Fig. 1-7B)ࠊPhos-tag ࢤࣝ࡟ࡼࡿゎᯒࢆ⾜ࡗࡓࠋࡑࡢ⤖ᯝࠊphos-tag ࢤࣝ୰࡟࠾࠸࡚ࠊFLJ00018 WT ࡜ẚ㍑ࡋࠊFLJ00018 T680A ࡢ EGF ่⃭࡟ࡼࡿ⛣ືᗘࢩࣇࢺࡀᙅࡵࡽࢀࡿࡇ࡜ࡀ᫂ࡽ࠿࡜ ࡞ࡗࡓ (Fig. 1-7C)ࠋࡇࢀࡽࡢࡇ࡜࠿ࡽࠊEGF ่⃭࡟ࡼࡗ࡚άᛶ໬ࡉࢀࡓ ERK ࡟ࡼࡗ࡚ FLJ00018 ࡢࢫࣞ࢜ࢽࣥ 680 ␒┠ࡀࣜࣥ㓟໬ࡉࢀࡿࡇ࡜ࡀ♧၀ࡉࢀࡓࠋࡋ࠿ࡋ࡞ࡀࡽࠊEGF ่ ⃭࡟క࠸ࠊFLJ00018 T680A ࡟ࡶࢃࡎ࠿࡟ṧࡿ⛣ືᗘࢩࣇࢺࡀࡳࡽࢀࠊࡇࡢࡇ࡜ࡣ FLJ00018

ࡢ680 ␒┠௨እࡢ࢔࣑ࣀ㓟㓄ิୖ࡟࠾࠸࡚ࠊEGF ่⃭࡟ࡼࡗ࡚ࣜࣥ㓟໬ࡉࢀࡿ࢔࣑ࣀ㓟ࡀᏑᅾ

ࡍࡿ࠿ࡶࡋࢀ࡞࠸ࡇ࡜ࢆ♧၀ࡋ࡚࠸ࡿࠋ

FLJ00018 ࡢ 680 ␒┠ࡢࢫࣞ࢜ࢽࣥṧᇶࡢࣜࣥ㓟໬ࡀ FLJ00018 ࡢάᛶ࡟୚࠼ࡿᙳ㡪࡟ࡘ࠸ ᳨࡚ウࢆࡍࡿⅭࠊSRE άᛶ ᐃࢆ⾜ࡗࡓࠋFigure 1-7D ࡟♧ࡍᵝ࡟ࠊFLJ00018 T680A ࡢ EGF

่⃭࡟ࡼࡿSRE άᛶୖ᪼ࡣࠊFLJ00018 WT ࡜ẚ࡭ኚ໬ࡀぢࡽࢀ࡞࠿ࡗࡓࠋࡲࡓࠊྛ✀ Rho ࡢ

ࢻ࣑ࢼࣥࢺࢿ࢞ࢸ࢕ࣈኚ␗యࢆ⏝࠸ࠊFLJ00018 T680A ࡀ RhoGTPase ࡟୚࠼ࡿᙳ㡪ࡢ᳨ウࢆ ⾜ࡗࡓࠋࡋ࠿ࡋ࡞ࡀࡽࠊFLJ00018 T680A ࡣࠊFLJ00018WT ࡜ྠᵝ࡟ (Fig. 1-3B) Rac, Cdc42 ࡟ᑐࡋ࡚άᛶࢆ᭷ࡋ࡚࠾ࡾࠊRhoGTPase ࡬୚࠼ࡿᙳ㡪࡟ࡘ࠸࡚ࡶኚ໬ࡀぢࡽࢀ࡞࠿ࡗࡓ (Fig. 1-7E)ࠋ

27

88. EGF ่⃭ࢆ௓ࡋࡓ FLJ00018 ࡟ࡼࡿ⚄⤒✺㉳ᙧᡂ࡜⣽⬊ఙᒎ

Fiig. 1--7 EGF ่่⃭࡟ࡼࡿFLJ000188ࣜࣥ㓟໬࡟㛵ࢃࡿ࢔࣑ࣀ㓟ࡢྠᐃ

A, B, NIH3T3 ⣽⬊ (A) ࡲࡓࡣ HEK293 ⣽⬊ (B) ࡟ EGFR, Myc ࢱࢢࢆ௜ຍࡋࡓ FLJ00018 ࡢⓎ⌧ ࣋ࢡࢱ࣮ࢆᅗ࡟♧ࡋࡓࡼ࠺࡟୍㐣ᛶ࡟ࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡋࡓࠋࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡋࡓ⣽⬊࡟ EGF (20 ng/ml) ࢆ⏝࠸࡚ 15 ศ㛫่⃭ࢆ⾜ࡗࡓࠋ➼㔞ࡢ⺮ⓑ㉁ࢆ 7.5% SDS-PAGE ࡛ศ㞳ࡋࡓࠋ࢖࣒ ࣀࣈࣟࢵࢺࡣࠊMyc ࢱࢢࡀ௜ຍࡉࢀࡓ FLJ00018 ࡢ᳨ฟࡢⅭ࡟ࠊAnti-Myc ᢠయࢆ⏝࠸ࡓࠋ

C, HEK293 ⣽⬊࡟ EGFR, Myc ࢱࢢࢆ௜ຍࡋࡓ FLJ00018 ࡢⓎ⌧࣋ࢡࢱ࣮ࢆᅗ࡟♧ࡋࡓࡼ࠺࡟୍㐣ᛶ

࡟ࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡋࡓࠋࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࢆ⾜ࡗࡓ⣽⬊࡟20 ng/ml ࡢ EGF ่⃭ (15 ศ㛫)

ࢆ⾜ࡗࡓࠋ➼㔞ࡢ⺮ⓑ㉁ࢆ13 PM Phos-tag AAL107, 26 PM MnCl2, 1.5%࢔࣮࢞ࣟࢫࢆຍ࠼ࡓ 3%

SDS-PAGE ࡛ศ㞳ࡋࡓࠋMyc ࢱࢢࡀ௜ຍࡉࢀࡓ FLJ00018 ࢆ᳨ฟࡍࡿⅭ࡟࢖࣒ࣀࣈࣟࢵࢺࡣ Anti-Myc ᢠయࢆ⏝࠸࡚⾜ࡗࡓࠋ

D, E, NIH3T3⣽⬊࡟ pSRE.L-luciferase, pRL-SV40 ࣉࣛࢫ࣑ࢻ DNA, FLJ00018, EGFR, pF4A-RhoA T19N, pF4A-Rac T17N, pF4A-Cdc42 T17N ࡢⓎ⌧࣋ࢡࢱ࣮ࢆࡑࢀࡒࢀ♧ࡋࡓࡼ࠺࡟ࢺࣛࣥࢫࣇ࢙ࢡ ࢩࣙࣥࡋࡓࠋࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡋࡓ⣽⬊࡟ࠊ20 ng/ml, 6 ᫬㛫ࡢ EGF ่⃭ࢆ⾜ࡗࡓࠋࣝࢩࣇ࢙ࣛ

࣮ࢮάᛶࡣdual-luciferase reporter assay system ࢆ⏝࠸࡚ ᐃࡋࠊᚓࡽࢀࡓάᛶࡣ Mock ࢆ 1.0 ࡜ࡋ

࡚ᶆ‽໬ࡋࡓ್ࢆ♧ࡋࡓࠋᐇ㦂ࡣᑡ࡞ࡃ࡜ࡶ3 ᅇ⾜࠸ࠊ್ࢆᖹᆒ್s S.D.࡛♧ࡋࡓࠋ

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

࠸ࡃࡘ࠿ࡢ RhoGEF ࡀ⚄⤒✺㉳ᙧᡂࡢࡼ࠺࡞⣽⬊ᙧែไᚚ࡟㛵୚ࡍࡿ࡜࠸࠺ሗ࿌ࡀ࠶ࡿ

(29,45)ࠋࡑࡇ࡛ࠊEGF ่⃭࡟ࡼࡿࠊFLJ00018 ࡢάᛶ໬ࡀ⚄⤒✺㉳ᙧᡂ࡟㛵୚ࡍࡿ࠿࡝࠺࠿࡟ ࡘ࠸᳨࡚ウࡋࡓࠋࡲࡎࠊNeuro-2a ⣽⬊࡟ FLJ00018, EGFR ࢆඹⓎ⌧ࡉࡏࠊEGF ่⃭ (20 ng/ml, 24 ᫬㛫) ࢆ⾜࠸ࠊ࢖࣒ࣀࣈࣟࢵࢺゎᯒ࡟ࡼࡿ FLJ00018 ࡢ⺮ⓑ㉁Ⓨ⌧ゎᯒࢆ⾜ࡗࡓࠋࡑࡢ⤖ᯝࠊ NIH3T3 ࡸ HEK293 ⣽⬊ྠᵝࠊEGF ่⃭࡟ࡼࡾ FLJ00018 ࡢ⛣ືᗘࢩࣇࢺࡀぢࡽࢀࡓ (ࢹ࣮ࢱ ࡟ࡣ♧ࡉ࡞࠸)ࠋࡑࡇ࡛ḟ࡟ࠊNeuro-2a ⣽⬊࡟༢㔞య࡛ാࡃ᪂つࡢ⺯ග⺮ⓑ㉁࢔ࢨ࣑ࢢ࣮ࣜࣥ (mAG) (32) ࠾ࡼࡧ mAG ࢱࢢ໬ࡋࡓ FLJ00018 (mAG-FLJ00018) ࢆ EGFR ࡜ඹⓎ⌧ࡉࡏࠊ⣽

⬊ᙧែኚ໬ࢆほᐹࡋࡓࠋࡑࡢ⤖ᯝࠊFLJ00018 WT ࡜ EGFR ࢆඹⓎ⌧ࡉࡏࡓ⣽⬊࡟࠾࠸࡚ࠊEGF

่⃭࡟ࡼࡾ⣽⬊ఙᒎ࡜⣽⬊✺㉳ᙧᡂࡀࡳࡽࢀࡓ (Fig. 1-8G, H)ࠋ୍᪉ࠊFLJ00018 ࣜࣥ㓟໬㒊఩

ࢆḞᦆࡉࡏࡓmAG-FLJ00018 T680A ࢆ EGFR ࡜ඹⓎ⌧ࡉࡏ࡚ࡶࠊFLJ00018 WT ࡛ぢࡽࢀࡓ

ᵝ࡞EGF ่⃭࡟ࡼࡿ⣽⬊ᙧែኚ໬ࡣᘬࡁ㉳ࡇࡉ࡞࠿ࡗࡓ (Fig. 1-8K, L)ࠋFLJ00018 ࡢࢫࣞ࢜

ࢽࣥ680 ␒┠ࡢࣜࣥ㓟໬ࡀ⣽⬊ᙧែኚ໬࡟㛵ࢃࡿࡇ࡜ࢆ☜࠿ࡵࡿⅭࠊࡑࢀࡒࢀࡢヨᩱ࡟ࡘ࠸࡚ࠊ

⏬ീゎᯒࢯࣇࢺࢆ౑⏝ࡋࡓ⤫ィゎᯒࢆ⾜ࡗࡓࠋFigure 1-8M ࡟♧ࡍᵝ࡟ࠊFLJ00018 WT ཬࡧ

F

Fig.. 1--88 EEGF ࢆࢆ௓ࡋࡓFFLJ00018 ࣜࣜࣥ㓟໬ࡣ⚄⤒✺㉳ఙ㛗࡜⣽⬊ᙧែኚ໬ࢆᘬࡁ㉳ࡇࡍࠋ

A-L, Neuro-2a ⣽⬊࡟༢㔞య࡛ാࡃ⺯ග⺮ⓑ㉁ mAG, mAG ࢆ௜ຍࡋࡓ FLJ00018 ࠾ࡼࡧ EGFR ࡢⓎ

⌧࣋ࢡࢱ࣮ࢆࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡋࡓࠋࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡢ3 ᫬㛫ᚋࠊ⣽⬊ࢆ 5% FBS ่࡛⃭ ࡋࠊ୍ᬌᇵ㣴ࡋࡓࠋ⩣᪥ࠊ⣽⬊ࢆ↓⾑Ύᇵᆅ࡟⨨ࡁ᥮࠼ࠊ20 ng/ml ࡢ EGF ่⃭ࢆ⾜ࡗࡓࠋ่⃭ 24 ᫬㛫ᚋࠊ⣽⬊ࢆ4% ࣃ࣒ࣛ࣍ࣝ࢔ࣝࢹࣄࢻᅛᐃࡋ⣽⬊ᙧែࡢほᐹࢆ⾜ࡗࡓࠋ M, ྛ sample ࠿ࡽࠊ↓సⅭ࡟㑅ᢥࡋࡓ 10 ᯛࡢ⺯ග⏬ീࢆྲྀᚓࡋࠊࡑࢀࡒࢀྜィ 50 ⣽⬊ࡢ⣽⬊࡟ࡘ ࠸࡚✺㉳ᵝࡢᵓ㐀ࡢ㛗ࡉࡢྜィࢆゎᯒࡋࡓࠋ⣽⬊ࡢィ ࡣImage J ࢯࣇࢺ࢙࢘࢔ࢆ⏝࠸࡚⾜ࡗࡓࠋ M

29

EGFR ࢆඹⓎ⌧ࡉࡏࡓ⣽⬊୰ࡢ⚄⤒✺㉳ᵝࡢᵓ㐀ࡢྜィࡢ㛗ࡉࡣࠊFLJ00018 T680A ࡜ EGFR ࢆඹⓎ⌧ࡉࡏࡓሙྜࡼࡾࠊ㛗࠸ࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡗࡓࠋࡇࢀࡽࡢ⤖ᯝࡼࡾࠊEGF ่⃭࡟ࡼࡿ FLJ00018 ࡢࢫࣞ࢜ࢽࣥ 680 ␒┠ࡢࣜࣥ㓟໬ࡣࠊEGF ่⃭࡟ࡼࡿ⚄⤒✺㉳ఙ㛗ࡸ⣽⬊ఙᒎ࡟㔜 せ࡛࠶ࡿࡇ࡜ࡀ♧၀ࡉࢀࡓࠋ

30

➨

➨

4 ⠇ ⪃ᐹ

 㐣ཤࠊFLJ00018 ࡀ୕㔞య G ⺮ⓑ㉁ࡢ GEJࢧࣈࣘࢽࢵࢺ࡜┤᥋⤖ྜࡍࡿࡇ࡜࡟ࡼࡾάᛶ໬ࡉ

ࢀࠊRhoGTPase ࡢ Rac1, Cdc42 ࢆάᛶ໬ࡍࡿࡇ࡜ࡀሗ࿌ࡉࢀࡓ (23)ࠋᮏ◊✲࡟࠾࠸࡚

FLJ00018 ࡣࠊE1AR ࢆ௓ࡋࡓ EGFR transactivation ࡟ࡼࡗ࡚ࡶάᛶ໬ࡉࢀࡿࡇ࡜ࢆ♧ࡋࡓ

(Fig. 1-2)ࠋࡇࢀࡲ࡛ endothelin, thrombin, calbachol, LPA ࢆྵࡴࠊ࠸ࡃࡘ࠿ࡢ GPCR ࢔ࢦࢽ

ࢫࢺࡀEGFR transactivation ࡟㛵ࢃࡿࡇ࡜ࡀ♧၀ࡉࢀ࡚࠸ࡿࠋࡲࡓࠊGPCR ࢆ௓ࡍࡿ EGFR

transactivation ࡢࢩࢢࢼࣝఏ㐩⤒㊰ࡣࠊ⣽⬊✀ࡸࠊ⣽⬊ࡀ࠾࠿ࢀࡿ⎔ቃ࡟ࡼࡗ࡚␗࡞ࡿࡇ࡜࠿ ࡽࠊᵝࠎ࡞ࢩࢢࢼࣝศᏊࡢ㛵୚ࡀᥦၐࡉࢀ࡚࠸ࡿࠋࡇࡢෆࠊ᭱ࡶゎ᫂ࡀ㐍ࢇ࡛࠸ࡿࢩࢢࢼࣝఏ㐩 ⤒㊰ࡣࠊADAM/heparin-binding EGF (HB-EGF) ࡟ࡼࡿ EGFR ࡢάᛶ໬ᶵᵓ࡛࠶ࡿࠋLPA ཷ ᐜయࡀࠊ࢚ࣥࢻࢪࢼࢫ࡞࡛ࣞ࣋ࣝⓎ⌧ࡋ࡚࠸ࡿሙྜࠊLPA ཷᐜయࡀ HB-EGF ౫Ꮡⓗ࡞ EGFR άᛶ໬ࢆ௓ࡋ࡚phospholipase CJ1 (PLCJ1) ཬࡧࠊPI3K/AKT ⤒㊰ࢆάᛶ໬ࡍࡿ (46)ࠋ୍᪉ࠊ E2AR ࡣࠊc-Src ཬࡧࠊEGFR ࡢάᛶ໬ࢆ௓ࡋ࡚ ERK ⤒㊰ࢆάᛶ໬ࡍࡿࡀࠊPLCJ1 ࡸࠊPI3K/AKT

⤒㊰ࡣάᛶ໬ࡉࢀ࡞࠸࡜࠸࠺ሗ࿌ࡶ࠶ࡿ (47)ࠋࡇࡢάᛶ໬ᶵᵓ࡟ຍ࠼ࠊEAR ่⃭ࢆ௓ࡍࡿ EGFR

transactivation ࡣࠊ࠸ࡃࡘ࠿◊✲ࢢ࣮ࣝࣉ࡟ࡼࡗ࡚ࠊ」ᩘࡢᶵᵓࢆ௓ࡋ࡚ᘬࡁ㉳ࡇࡉࢀ࡚࠸ࡿ

࡜࠸࠺ࡇ࡜ࡀᥦၐࡉࢀ࡚࠸ࡿࠋDakka ࡽࡣࠊE2AR ࢦࢽࢫࢺ่⃭࡟ࡼࡾ Gs ࢩࢢࢼࣝ࠿ࡽ Gi ࢩ

ࢢࢼࣝ࡬ࡢኚ᥮ࡀ⏕ࡌࠊGi ࢩࢢࢼࣝࡀ GEJ, Src ࢆ௓ࡋ࡚ Ras/MAPK ⤒㊰ࡀάᛶ໬ࡉࡏࡿࡇ࡜ ࢆぢฟࡋ࡚࠸ࡿ (48)ࠋࡋ࠿ࡋ࡞ࡀࡽࠊ௚ࡢࢢ࣮ࣝࣉ࡟ࡼࡗ࡚ࠊGs/Gi ࢫ࢖ࢵࢳࣥࢢ㠀౫Ꮡⓗ࡟ Src ࢆ௓ࡋ࡚ EGFR/MAPK ࡢάᛶ໬ࡀ⏕ࡌࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿ (49)ࠋࡉࡽ࡟ࠊGRK5 ࠾ࡼ ࡧࠊGRK6/E-arrestin/Src/EGFR ࡜࠸࠺⤒㊰ࢆ௓ࡍࡿ transactivation ᶵᵓࡢᏑᅾࡶ♧၀ࡉࢀ࡚ ࠸ࡿ (40)ࠋࡇࢀࡽࡢሗ࿌ࡣࠊEAR ࢆ௓ࡋ࡚ EGFR transactivation ࡟ࡼࡗ࡚ Ras-MAPK ⤒㊰ࡀ

άᛶ໬ࡉࢀࡿ࡜࠸࠺௒ᅇࡢⓎぢ࡜୍⮴ࡋ࡚࠾ࡾࠊFLJ00018 ࡀEAR ࢆ௓ࡋ࡚άᛶ໬ࡉࢀࡓ

EGFR/Ras/MAPK ࡟ࡼࡗ࡚ࣜࣥ㓟໬ࡉࢀࡿ⤒㊰ࡢᏑᅾࡢྍ⬟ᛶ࡜▩┪ࡋ࡞࠸ࠋࡋ࠿ࡋ࡞ࡀࡽࠊ

ᮏ◊✲࡛ࡣࠊE1AR ࡢୗὶ࡟࠾࠸࡚ࠊ࡝ࡢࡼ࠺࡞⤒㊰ࢆ௓ࡋࡓ EGFR ࡢ transactivation ࡀ㉳ࡁ

࡚࠸ࡿࡢ࠿ࠊヲ⣽ࢆ᳨ウࡍࡿࡇ࡜ࡀ࡛ࡁ࡞࠿ࡗࡓࠋ

Vascular smooth muscle ⣽⬊࡟࠾࠸࡚ࠊThrombin ࡟ࡼࡿ protease-activated receptors 1 (PAR1) ࡢάᛶ໬ࢆ௓ࡋࡓ EGFR transactivation ࡀ⏕ࡌࡿࠋࡇࡢ Thrombin ࡟ࡼࡿ EGFR ࡢά

31

ᛶ໬࡟ࡼࡾࠊRhoA ࡀάᛶ໬ࡉࢀࡿ (50)ࠋࡇࡢࡇ࡜ࡣࠊEGFR transactivation ౫Ꮡⓗ࡞⤒㊰ࢆ

௓ࡋ࡚RhoGEF ࡀάᛶ໬ࡉࢀࡿྍ⬟ᛶࢆ♧၀ࡋ࡚࠸ࡿࠋࡋ࠿ࡋ࡞ࡀࡽࠊE2AR /Gi ่⃭࡟ࡼࡗ࡚ࠊ

Src ࢆ௓ࡋ࡚⾑ᑠᯈ⏤᮶ቑṪᅉᏊཷᐜయ (PDGFR)/PI3K ࢩࢢࢼࣝࡀάᛶ໬ࡉࢀࡿ࡜࠸࠺ሗ࿌ࡶ

࠶ࡿ (39)ࠋࡉࡽ࡟ࠊE1AR ࡜ EGFR ࡢ┦஫స⏝࡬ࡢE-࢔ࣞࢫࢳࣥࡢ⤖ྜࡀࠊEGFR ࡢ⊂❧ࡋࡓ

άᛶ໬࡜EGFR transactivation ࡟ࡼࡿάᛶ໬࡟࠾ࡅࡿࢩࢢࢼࣝࡢ㐪࠸ࢆ⏕ࡌࡉࡏ࡚࠸ࡿ࡜࠸࠺

ሗ࿌ࡀ࠶ࡿ (42)ࠋᐇ㝿ࠊᮏ◊✲࡟࠾࠸࡚ࠊFLJ00018, E1AR ཬࡧE-ARKct ࢆඹⓎ⌧ࡉࡏࡓሙྜࠊ

E1AR ่⃭࡟ࡼࡿ SRE άᛶࡀ᏶඲࡟ࡣᢚไࡉࢀ࡞࠿ࡗࡓࠋࡲࡓࠊFLJ00018 T680A ࡢ㟁ẼὋື

ୖࡢ⛣ືᗘࢩࣇࢺࡣࠊFLJ00018 WT ࡟ẚ࡭ࠊ᏶඲࡟ࡣᢚไࡉࢀࡎࠊSRE άᛶ࡟ࡶኚ໬ࡀ⏕ࡌ

࡞࠿ࡗࡓࡇ࡜࠿ࡽࠊE1AR ่⃭᫬࡟ EGFR transactivation ࢆྵࡴ」ᩘࡢࢩࢢࢼࣝࡀྠ᫬࡟ാ࠸

࡚࠸ࡿࡇ࡜ࡶ⪃࠼ࡽࢀࡿࠋ

 ᮏ◊✲࡛ࡣࡲࡓࠊFLJ00018 ࡀ EGFR ࡢ┤᥋่⃭࡟ࡼࡗ࡚ࡶάᛶ໬ࡉࢀࡿࡇ࡜ࢆ♧ࡋࡓ (Fig.

1-3)ࠋEGF ่⃭࡟ࡼࡿ FLJ00018 ࡢάᛶ໬࡟ࡣࠊRas-MAPK ⤒㊰ࡀ㛵ࢃࡗ࡚࠾ࡾ (Fig. 1-4, 1-5)ࠊ

ࡇࡢ⤒㊰࡟ࡼࡗ࡚FLJ00018 ࡀࣜࣥ㓟໬ࢆཷࡅࡿࡇ࡜ࢆ♧ࡋࡓ (Fig. 1-5)ࠋEGFR ࡟㝈ࡽࡎࠊቑ

ṪᅉᏊཷᐜయࡸ⚄⤒ᡂ㛗ᅉᏊཷᐜయࡢୗὶ࡟࠾࠸࡚ࣜࣥ㓟໬ࡉࢀࡿRhoGEF ࡣࡇࢀࡲ࡛࡟࠸ࡃ

ࡘ࠿ぢ࠸ࡔࡉࢀ࡚࠸ࡿ (27)ࠋࡓ࡜࠼ࡤࠊRhoGEF ࡢ୍✀ࠊAsef ࡣࠊEGFR ࢩࢢࢼࣝࡢୗὶ࡟࠾

࠸࡚Src ࣇ࢓࣑࣮ࣜ࢟ࢼ࣮ࢮ౫Ꮡⓗ࡞⤒㊰࡟ࡼࡗ࡚ Tyr 94 ࡀࣜࣥ㓟໬ࡉࢀࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚

࠸ࡿ (28)ࠋࣜࣥ㓟໬ࡉࢀࡓ Asef ࡣ Rac1, Cdc42 ࢆάᛶ໬ࡍࡿࠋ୍᪉ࠊูࡢ RhoGEF ࡛࠶ࡿEPIX ࡣࠊFGFR ࡢࢩࢢࢼࣝࡢୗὶ࡛ാࡃ Ras-ERK-Pak2 ⤒㊰࡟ࡼࡗ࡚ࠊSer 525 ࡜ Thr 526 ࡀࣜࣥ 㓟໬ࢆ࠺ࡅࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿ (29)ࠋࣜࣥ㓟໬ࡉࢀࡓEPIX ࡣ PC12 ⣽⬊ෆ࡛⚄⤒✺㉳ఙ㛗

ࢆᘬࡁ㉳ࡇࡍࠋEPIX ࡣࡉࡽ࡟ࠊEGFR ࡢୗὶ࡟࠾࠸࡚ࡶࣜࣥ㓟໬ࡉࢀࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠾ࡾࠊ

ࡇࡢࣜࣥ㓟໬࡟ࡣSrc ࣇ࢓࣑࣮ࣜ࢟ࢼ࣮ࢮ࡟ࡼࡿ Tyr 442 ࡢࣜࣥ㓟໬ࡀ㛵୚ࡋ࡚࠸ࡿ (51)ࠋቑ

ṪᅉᏊཷᐜయࢩࢢࢼࣝࡢୗὶࡢศᏊ࡟ࡼࡿRhoGEF ࡢࣜࣥ㓟໬ࡣ Rac1, Cdc42 ࡟ᑐࡍࡿ GEF

࡟ຍ࠼ࠊRhoA ࡟ᑐࡍࡿ GEF ࡛࠶ࡿ GEF-H1 ࡶ ERK1/2 ࡟ࡼࡾࠊThr 678 ࡀࣜࣥ㓟໬ࡉࢀࡿ (52)ࠋࡇࡢࡼ࠺࡟ RhoGEF ࡀቑṪᅉᏊཷᐜయࡸ⚄⤒ᰤ㣴ᅉᏊཷᐜయ࡟ࡼࡗ࡚άᛶ໬ࡉࢀࡿ࡜࠸

࠺ሗ࿌ࡣ」ᩘ࡞ࡉࢀ࡚࠾ࡾࠊRhoGEF ࡢάᛶไᚚࡣ GPCR ࡔࡅ࡛࡞ࡃࠊEGFR ࢩࢢࢼࣝࡢᵝ࡞

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࠸ࡃࡘ࠿ࡢRhoGEF ࡢάᛶ໬࡟ࡼࡾ⚄⤒✺㉳ఙ㛗ࡀᘬࡁ㉳ࡇࡉࢀࡿࡇ࡜ࡀ᫂ࡽ࠿࡟࡞ࡾࡘࡘ

࠶ࡿࠋTiam1 ࡣࠊ⬻࡛ᶵ⬟ࡋ࡚࠸ࡿ⚄⤒ᡂ㛗ᅉᏊཷᐜయ TrkA ࡸ TrkB ࡟ࡼࡗ࡚άᛶ໬ࡉࢀࡿࠋ NGF ࡟ࡼࡿ TrkA ࡢάᛶ໬࡟⥆ࡁࠊTiam1 ࡀ Ras ࡜⤖ྜࡋࠊࡑࡢୗὶ࡟࠾࠸࡚ Rac ࡢάᛶ໬ ࢆ௓ࡋࡓ⚄⤒✺㉳ఙ㛗ࡀᘬࡁ㉳ࡇࡉࢀࡿ (53)ࠋࡲࡓࠊBurridge ࡽࡢࢢ࣮ࣝࣉࡣࠊEGF ่⃭࡟

ࡼࡗ࡚RhoG ࡀάᛶ໬ࡍࡿࡇ࡜ࢆ♧ࡋࠊRhoG ࡢάᛶ໬࡟ࡣࠊVav ࣇ࢓࣑࣮ࣜRhoGEF ཬࡧࠊ

RhoG ≉␗ⓗ RhoGEF ࡢ PLEKHG6 ࡀ㛵୚ࡍࡿࡇ࡜ࡶྜࢃࡏ࡚ሗ࿌ࡋ࡚࠸ࡿ (54)ࠋࡲࡓࠊู ࡢㄽᩥ࡛ࡣࠊRhoGEF ࡢ୍✀ࠊTrio ࡀࠊRhoG ౫Ꮡⓗ࡞⤒㊰ࢆ௓ࡋ࡚⚄⤒✺㉳ఙ㛗࡟㛵୚ࡍࡿ ࡇ࡜ࡀ♧ࡉࢀ࡚࠸ࡿ (55)ࠋࡇࢀࡽࡢሗ࿌࡜୍⮴ࡋ࡚ࠊᮏ◊✲࡟࠾࠸࡚ࡶࠊRas/MAPK ⤒㊰࡟ࡼ ࡗ࡚ࣜࣥ㓟໬ࡉࢀࡓ FLJ00018 ࡀࠊ⚄⤒✺㉳ࡢఙ㛗࡟㛵ࢃࡗ࡚࠸ࡿࡇ࡜ࢆ♧ࡋࡓࠋFLJ00018 ࡣ⏕యෆ࡟ᗈࡃⓎ⌧ࡀࡳࡽࢀࠊ⬻࡟࠾࠸࡚ࡶࡑࡢⓎ⌧ࡀぢࡽࢀࡿࡇ࡜࠿ࡽࠊ௒ᅇぢࡽࢀࡓࡇࡢ FLJ00018 ࡢࣜࣥ㓟໬࡟ࡼࡿ✺㉳ఙ㛗ࡣࠊ⏕యෆ࡛ࡶ⏕ࡌ࡚࠸ࡿ⌧㇟࡞ࡢ࠿ࡶࡋࢀ࡞࠸ࠋࡋ࠿ࡋ ࡞ࡀࡽࠊඛ࡟ࡶ㏙࡭ࡓࡼ࠺࡟ࠊEGF ࢩࢢࢼࣝ࡟ࡼࡿࣜࣥ㓟໬㒊఩ࢆኚ␗ࡉࡏࡓ FLJ00018 T680A ࡣࠊ✺㉳ఙ㛗ࡣぢࡽࢀ࡞ࡃ࡞ࡿࡶࡢࡢࠊRhoGTPase ࡟ᑐࡍࡿάᛶ࡟ኚ໬ࡣぢࡽࢀ࡞࠿ࡗࡓࠋ680 ␒┠ࡢࣜࣥ㓟໬ࡣࠊ┤᥋ⓗ࡞ Rho ࡟ᑐࡍࡿάᛶ࡛ࡣ࡞ࡃࠊ⣽⬊ෆᒁᅾࡸᵓ㐀ࡢኚ໬࡟㛵ࢃࡗ࡚ ࠸ࡿྍ⬟ᛶࡶ࠶ࡾࠊ௒ᚋヲ⣽࡞᳨ウ௒ᚋヲ⣽࡞᳨ウࡀᚲせ࡜⪃࠼ࡽࢀࡓࠋࡲࡓࠊඛࡢEPIX ࡢ౛ ࡟ࡳࡽࢀࡿࡼ࠺࡟ࠊࢩࢢࢼࣝఏ㐩⤒㊰ࡢ✀㢮࡟ࡼࡾࠊࣜࣥ㓟໬ࡉࢀࡿ࢔࣑ࣀ㓟ṧᇶࡣ␗࡞ࡗ࡚࠾ ࡾࠊ୍ࡘࡢRhoGEF ศᏊࡣ」ᩘࡢࣜࣥ㓟໬ࡉࢀ࠺ࡿ࢔࣑ࣀ㓟ṧᇶࢆᣢࡗ࡚࠸ࡿࡇ࡜ࡀண᝿ࡉࢀ ࡿࠋࡇࡢࡇ࡜ࡣࠊࣜࣥ㓟໬ࡉࢀࡿ࢔࣑ࣀ㓟ṧᇶࡢ఩⨨࡟ࡼࡾࠊୗὶࡢࢩࢢࢼࣝఏ㐩⤒㊰ࡀ␗࡞ࡿ ྍ⬟ᛶࢆ♧၀ࡋ࡚࠸ࡿࠋᮏ◊✲࡛ぢ࠸ࡔࡉࢀࡓEGF ࢩࢢࢼࣝ࡟ࡼࡿ FLJ00018 ࡢࣜࣥ㓟໬ࡶࠊ ࣜࣥ㓟໬㒊఩࡟ࡼࡾࠊ㑇ఏᏊ㌿෗άᛶ࡟㛵ࢃࡿࣜࣥ㓟໬ࡸ⣽⬊ᒁᅾࡢኚ໬࡟㛵ࢃࡿࣜࣥ㓟໬➼ࠊ ୗὶࡢࢩࢢࢼࣝఏ㐩⤒㊰ࡸ⣽⬊ᶵ⬟࡬୚࠼ࡿᙺ๭ࡶ␗࡞ࡗ࡚࠸ࡿࡢ࠿ࡶࡋࢀ࡞࠸ࠋ ௒ᚋࠊ FLJ00018 ࡢࣜࣥ㓟໬ࡸࣜࣥ㓟໬ࡢ㒊఩࡟ࡼࡿࢩࢢࢼࣝఏ㐩ᶵᵓࡢ㐪࠸ࡢヲ⣽ࢆゎ᫂ࡍࡿࡇ࡜ ࡟ࡼࡾࠊ࢔ࢡࢳࣥ⣽⬊㦵᱁෌ᵓ⠏ᶵᵓࡸࡑࡢ◚⥢࡟࡜ࡶ࡞࠺ࡀࢇࡸ⚄⤒⑌ᝈࡢゎ᫂࡟ᙺ❧ࡘ࡜⪃ ࠼ࡽࡿࠋ

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F

Fiig. 1--99 ᮏᮏ◊✲࡛ぢ࠸ࡔࡉࢀࡓEEGFR ttransactivation ࢆࢆ௓ࡋࡓFFLJ00018 ࡢࡢࣜࣥ㓟໬࡜άᛶ໬ᶵᵓࠋ

E1AR ่⃭࡟ࡼࡾ EGFR transactivation ࡀㄏᑟࡉࢀࡿࠋάᛶ໬ࡉࢀࡓ EGFR ࡣࠊMAPK ࢝ࢫࢣ࣮ࢻࢆ

௓ࡋ࡚ࠊFLJ00018 ࡢࣜࣥ㓟໬ࢆᘬࡁ㉳ࡇࡍࠋFLJ00018 ࡣࠊMAPK ⤒㊰࡟࠾ࡅࡿ ERK1/2 ࡟ࡼࡗ࡚ 680 ␒┠ࡢࢫࣞ࢜ࢽࣥṧᇶ࡟ࣜࣥ㓟໬ࢆཷࡅࡿࠋFLJ00018 ࡣࡉࡽ࡟ࠊMAPK ࡶࡋࡃࡣ MEK ࡢୗὶࡢ௚ࡢ ศᏊ࡟ࡼࡗ࡚ 465-615 ␒┠ࠊ965-1386 ␒┠ࡢࢭࣜࣥ/ࢫࣞ࢜ࢽࣥṧᇶࡶྠ᫬࡟ࣜࣥ㓟໬ࢆཷࡅࡿྍ⬟ᛶ ࡀ♧၀ࡉࢀࡓࠋࣜࣥ㓟໬ࡉࢀࡓFLJ00018 ࡣ RhoGTPase ࡢ Cdc42, Rac1 ࢆάᛶ໬ࡋࠊ⣽⬊ᙧែኚ໬࡜ ⚄⤒✺㉳⏕ᡂࢆᘬࡁ㉳ࡇࡍࠋ J D E E1AR transactivation EGFR Isoproterenol H-Ras MEK MAPK UNKNOWN cytoskeltal regulation DH PH T680 Cdc42䚸Rac activation FLJ00018 UNKNOWN