Extracellular Phosphorylation of Collagen XVII by Ecto-Casein Kinase 2 Inhibits Ectodomain Shedding*
Extracellular Phosphorylation of Collagen XVII by Ecto-Casein Kinase 2 Inhibits Ectodomain Shedding*
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胞外酪蛋白激酶 2 对 XVII 胶原蛋白的胞外磷酸化可抑制胞外域脱落*
DOI：
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发表时间：
2007
影响因子：
4.8
通讯作者：
L. Bruckner
中科院分区：
文献类型：
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作者：
E. Zimina;A. Fritsch;B. Schermer;A. Bakulina;M. Bashkurov;T. Benzing;L. Bruckner
细胞外磷酸化是调节细胞配体相互作用和信号转导的重要机制。在这里，我们表明，细胞表面受体胶原蛋白XVII的细胞外磷酸化调节脱落的胞外域。胶原XVII是胶原跨膜蛋白新家族的成员，是半桥粒的组分，介导皮肤中表皮与真皮的粘附。胞外结构域通过ADAM（一种去整合素和金属蛋白酶）家族的金属蛋白酶，主要是肿瘤坏死因子-α转化酶（TACE）从细胞表面组成性脱落。我们使用生物化学，诱变和结构建模方法来描述控制胞外域切割的机制。细胞外磷酸化的标准测定，用细胞不可渗透的[γ-32 P]ATP孵育完整的角质形成细胞，导致胶原XVII标记。广谱细胞外激酶抑制剂K252 b和特异性酪蛋白激酶2（CK 2）抑制剂均显著降低了这一点。胶原蛋白XVII肽含有一个假定的CK 2识别位点磷酸化的CK 2在体外，披露Ser 542和Ser 544在胞外域作为磷酸基团受体。Ser 544在体内和体外的磷酸化通过用磷酸化表位特异性抗体对表皮和HaCaT角质形成细胞提取物进行免疫印迹来证实。在功能上，CK 2激酶活性的抑制或磷酸化受体Ser 544突变为Ala显著增加胞外结构域脱落，而CK 2 α的过表达抑制胶原XVII的切割。结构建模表明丝氨酸残基的磷酸化阻止了TACE与其底物的结合。因此，胞外CK 2对胶原XVII的胞外磷酸化抑制了TACE对其的脱落，代表了调节上皮细胞粘附和运动的新机制。
Ecto-phosphorylation is emerging as an important mechanism to regulate cellular ligand interactions and signal transduction. Here we show that extracellular phosphorylation of the cell surface receptor collagen XVII regulates shedding of its ectodomain. Collagen XVII, a member of the novel family of collagenous transmembrane proteins and component of the hemidesmosomes, mediates adhesion of the epidermis to the dermis in the skin. The ectodomain is constitutively shed from the cell surface by metalloproteinases of the ADAM (a disintegrin and metalloproteinase) family, mainly by tumor necrosis factor-α converting enzyme (TACE). We used biochemical, mutagenesis, and structural modeling approaches to delineate mechanisms controlling ectodomain cleavage. A standard assay for extracellular phosphorylation, incubation of intact keratinocytes with cell-impermeable [γ-32P]ATP, led to collagen XVII labeling. This was significantly diminished by both broad-spectrum extracellular kinase inhibitor K252b and a specific casein kinase 2 (CK2) inhibitor. Collagen XVII peptides containing a putative CK2 recognition site were phosphorylated by CK2 in vitro, disclosing Ser542 and Ser544 in the ectodomain as phosphate group acceptors. Phosphorylation of Ser544 in vivo and in vitro was confirmed by immunoblotting of epidermis and HaCaT keratinocyte extracts with phosphoepitope-specific antibodies. Functionally, inhibition of CK2 kinase activity or mutation of the phosphorylation acceptor Ser544 to Ala significantly increased ectodomain shedding, whereas overexpression of CK2α inhibited cleavage of collagen XVII. Structural modeling suggested that the phosphorylation of serine residues prevents binding of TACE to its substrate. Thus, extracellular phosphorylation of collagen XVII by ecto-CK2 inhibits its shedding by TACE and represents novel mechanism to regulate adhesion and motility of epithelial cells.
影响因子：
2.9
作者：
Hu, X;Balaz, S;Shelver, WH
通讯作者：
Shelver, WH
影响因子：
15.9
作者：
PILLAI, S;BIKLE, DD
通讯作者：
BIKLE, DD
影响因子：
2.9
作者：
Hu, X;Shelver, WH
通讯作者：
Shelver, WH