Protein Kinase C (cid:1) Regulates Function of the DF3/MUC1 Carcinoma Antigen in (cid:2) -Catenin Signaling*
Protein Kinase C (cid:1) Regulates Function of the DF3/MUC1 Carcinoma Antigen in (cid:2) -Catenin Signaling*
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蛋白激酶 C (cid:1) 调节 (cid:2) -连环蛋白信号传导* 中 DF3/MUC1 癌抗原的功能
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通讯作者：
John Cottrell
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作者：
M. Bartlet;W. Jeffery;Hans F. Hansen;Darryl J. Pappin;John Cottrell
DF 3/MUC 1粘蛋白样糖蛋白在大多数人类肿瘤中异常过表达。MUC 1胞质结构域直接与（cid：2）-连环蛋白相互作用，连环蛋白是哺乳动物上皮细胞粘附连接的组分。本结果表明MUC 1与蛋白激酶C（cid：1）（PKC（cid：1））缔合。邻近MUC 1胞质结构域中的（cid：2）-连环蛋白结合基序的TDR序列作为PKC（cid：1）磷酸化的位点发挥作用。我们发现，PKC（cid：1）对MUC 1的磷酸化增加了MUC 1和（cid：2）-cate-nin在体外和体内的结合。MUC 1-PKC（cid：1）相互作用的功能意义进一步得到以下证明的支持：PKC（cid：1）磷酸化位点的突变消除了MUC 1介导的（cid：2）-连环蛋白与E-钙粘蛋白结合的降低。我们还发现，MUC 1对锚定非依赖性生长的刺激作用被PKC（cid：1）磷酸化位点的突变所消除。这些发现支持PKC（cid：1）在调节MUC 1和（cid：2）-连环蛋白信号通路之间的相互作用中的新作用。将蛋白激酶C野生型MUC 1/CD和MUC 1/CD（T41 A）与0.3（cid：9）g重组PKC（cid：4）（PanVera）在4 ℃下孵育1小时。通过用抗MUC 1/CD（20）和抗PKC（cid：4）进行免疫印迹来分析抗PKC（cid：4）免疫沉淀物。在其他实验中，将纯化的His标记的野生型和突变体MUC 1/CD蛋白与0.3（cid：9）g PKC（cid：4）（PanVera）在不存在和存在200（cid：9）M ATP的情况下在30 ℃下孵育20分钟。然后加入与谷胱甘肽珠结合的GST-（cid：2）-连环蛋白，并将反应物在4°C温育1小时。用抗MUC 1/CD对沉淀的蛋白进行免疫印迹分析（20）。体外磷酸化-将纯化的His标记的野生型和突变型MUC 1/CD蛋白与0.3（cid：9）g重组PKC（cid：4）（PanVera）在激酶缓冲液（20 mM Tris-HCl，pH 7.6，10 mM MgCl 2，5（cid：9）M二硫苏糖醇）中孵育。通过加入10（cid：9）Ci [（cid：3）-32 P]ATP引发反应，并在30 °C下孵育20分钟。通过SDS-PAGE分离磷酸化蛋白并通过放射自显影进行分析。将细胞置于补充有胎血清抗生素的改良Eagle培养基中。将细胞悬浮液在60 mm培养皿中的3.5 ml琼脂/Dulbecco改良Eagle培养基上分层。一
The DF3/MUC1 mucin-like glycoprotein is aberrantly overexpressed in most human carcinomas. The MUC1 cytoplasmic domain interacts directly with (cid:2) -catenin, a component of the adherens junction of mammalian epithelial cells. The present results demonstrate that MUC1 associates with protein kinase C (cid:1) (PKC (cid:1) ). A TDR sequence adjacent to the (cid:2) -catenin binding motif in the MUC1 cytoplasmic domain functions as a site for PKC (cid:1) phosphorylation. We show that phosphorylation of MUC1 by PKC (cid:1) increases binding of MUC1 and (cid:2) -cate-nin in vitro and in vivo . The functional significance of the MUC1-PKC (cid:1) interaction is further supported by the demonstration that mutation of the PKC (cid:1) phosphorylation site abrogates MUC1-mediated decreases in binding of (cid:2) -catenin to E-cadherin. We also show that the stim-ulatory effects of MUC1 on anchorage-independent growth are abrogated by mutation of the PKC (cid:1) phosphorylation site. These findings support a novel role for PKC (cid:1) in regulating the interaction between MUC1 and the (cid:2) -catenin signaling pathway. The protein kinase C wild-type MUC1/CD and MUC1/CD(T41A) were incubated with 0.3 (cid:9) g of recombinant PKC (cid:4) (PanVera) for 1 h at 4 °C. Anti-PKC (cid:4) immunoprecipitates were analyzed by immunoblotting with anti-MUC1/CD (20) and anti-PKC (cid:4) . In other experiments, purified His- tagged wild-type and mutant MUC1/CD proteins were incubated with 0.3 (cid:9) g of PKC (cid:4) (PanVera) in the absence and presence of 200 (cid:9) M ATP for 20 min at 30 °C. GST- (cid:2) -catenin bound to glutathione beads was then added, and the reaction was incubated fo r 1 h at 4°C. The precipitated proteins were subjected to immunoblot analysis with anti-MUC1/CD (20). In Vitro Phosphorylation— Purified His-tagged, wild-type and mu- tant MUC1/CD proteins were incubated with 0.3 (cid:9) g of recombinant PKC (cid:4) (PanVera) in kinase buffer (20 m M Tris-HCl, pH 7.6, 10 m M MgCl 2 , 5 (cid:9) M dithiothreitol). The reactions were initiated by addition of 10 (cid:9) Ci of [ (cid:3) - 32 P]ATP and incubated for 20 min at 30 °C. Phosphorylated proteins were separated by SDS-PAGE and analyzed by autoradiography. Cells were in modified Eagle’s medium supplemented fetal serum antibiotics. The cell suspension was layered over 3.5 ml agar/Dulbecco’s modified Eagle’s medium in 60-mm dishes. One