Protein kinase C substrates that drive motility of cancer cells.

機譯：驅動癌細胞運動的蛋白激酶C底物。

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As the intracellular receptor of tumor promoting phorbol esters, protein kinase C (PKC) is functionally linked to carcinogenesis and metastasis. Therefore, it is crucial to identify substrates of PKC in order to understand the mechanisms by which these substrate proteins participate in cancer-related phenotypes such as motile behavior. The work to be described consists of two projects: (1) new PKC substrates that contribute to the motility phenotype of human breast cells, and (2) the role of a known PKC substrate, MARCKS (Myristoylated Alanine- Rich C-Kinase Substrate) in the motility pathway of mouse melanoma cells.;To identify direct substrates, a chemical-genetic approach was used to engineer the ATP binding site of PKC-alpha, delta and &zgr; to enable them to bind an unnatural ATP analogue. Consequently, phosphorylation is attributed exclusively to the mutant enzyme, and the phosphorylated protein bands are potential substrates. Following expression in human breast cells (MCF-10A), co-immunoprecipitation of the mutant enzyme bound with high affinity protein substrates was carried out. Following addition of an ATP analogue, a number of phosphorylated proteins were produced and subsequently analyzed by mass spectrometry. PKC-alpha and delta traceable kinases had very similar phosphorylation patterns, whereas the profile for PKC-&zgr; was distinct. Several potential protein substrates involved in cytoskeletal structure were identified for PKC-alpha and PKC-delta, including proteins that bind small GTP-binding proteins (Rho Kinase-1, Cdc42ep4, Rho/cdc42/Rac activating protein-1), as well as proteins that had been previously documented as PKC substrates (IQGAP, VASP).;In a second project, the role of MARCKS was investigated in the context of mouse melanoma cell motility. As a protein that is known to crosslink actin bundles to the plasma membrane, this PKC substrate has been linked with cell migration and adhesion. Upon phosphorylation by PKC, MARCKS is released into the cytoplasm, thereby promoting actin rearrangement. Weakly metastatic B16 F1 cells do not express detectable phospho-MARCKS. However, F1 cells that are engineered to express a pseudo-phosphorylated mutant of MARCKS exhibit elevated motility. When F1 cells are treated with okadaic acid (OA), an inhibitor of protein phosphatases, increases in both phospho-MARCKS and motility are observed. OA-induced motility can be substantially eliminated when cells are pre-treated with a shRNA reagent to knock down MARCKS expression, or when expressing a phosphorylation-resistant mutant of MARCKS. Furthermore, a phosphorylation-resistant MARCKS mutant that was made exclusively cytoplasmic due to lack of a myristoyl group was observed to inhibit motility of OA-treated F1 cells as well as motility of highly metastatic melanoma cells (mouse F10 and human A375 cells). These findings imply that MARCKS promotes motility through cytoplasmic interactions involving its phosphorylated effector domain. It is concluded that phospho-MARCKS has a previously undocumented role in the cytoplasm that promotes motile behavior and possibly the metastatic potential of many different cancer cells.;Overall, this work describes substrates of PKC that transmit the motility signal in cancer cells, and suggests novel targets for the development of anti-metastasis agents.

機譯：作為腫瘤促進佛波酯的細胞內受體，蛋白激酶C（PKC）在功能上與致癌和轉移有關。因此，至關重要的是鑒定PKC的底物，以了解這些底物蛋白參與癌癥相關表型（如運動行為）的機制。待描述的工作包括兩個項目：（1）有助于人類乳腺癌細胞運動表型的新PKC底物，以及（2）已知PKC底物MARCKS（富含Myristoylated丙氨酸的C激酶底物）的作用。為了鑒定直接底物，使用化學遺傳學方法對PKC-α，δ和＆zgr的ATP結合位點進行工程改造。使它們能夠結合非天然的ATP類似物。因此，磷酸化僅歸因于突變酶，而磷酸化的蛋白帶是潛在的底物。在人乳腺細胞（MCF-10A）中表達后，對與高親和力蛋白質底物結合的突變酶進行了免疫共沉淀。加入ATP類似物后，產生了許多磷酸化蛋白，隨后通過質譜分析。 PKC-α和δ可追蹤的激酶具有非常相似的磷酸化模式，而PKC-＆zgr的分布則為磷酸化。是與眾不同的。 PKC-alpha和PKC-delta的幾種可能的蛋白質底物都已被發現，包括與小GTP結合蛋白結合的蛋白（Rho Kinase-1，Cdc42ep4，Rho / cdc42 / Rac活化蛋白-1）以及以前已被證明是PKC底物的蛋白質（IQGAP，VASP）。；在第二個項目中，研究了MARCKS在小鼠黑色素瘤細胞運動中的作用。作為已知將肌動蛋白束交聯到質膜的蛋白質，這種PKC底物與細胞遷移和粘附有關。通過PKC磷酸化后，MARCKS釋放到細胞質中，從而促進肌動蛋白重排。轉移性弱的B16 F1細胞不表達可檢測的磷酸化MARCKS。但是，被工程化以表達MARCKS假磷酸化突變體的F1細胞表現出提高的運動能力。當F1細胞用蛋白磷酸酶的抑制劑岡田酸（OA）處理時，可觀察到磷酸MARCKS和運動性均增加。當用shRNA試劑預處理細胞以降低MARCKS的表達，或表達抗磷酸化的MARCKS突變體時，可以基本上消除OA誘導的運動。此外，觀察到由于缺乏肉豆蔻酰基而僅具有胞質性的抗磷酸化的MARCKS突變體抑制了OA處理的F1細胞的運動以及高度轉移性黑素瘤細胞（小鼠F10和人A375細胞）的運動。這些發現暗示MARCKS通過涉及其磷酸化的效應子結構域的細胞質相互作用促進運動。結論是，磷酸-MARCKS在細胞質中具有以前未記載的作用，可促進許多不同癌細胞的運動行為以及可能的轉移潛能。總體而言，這項工作描述了在癌細胞中傳遞運動信號的PKC底物，并暗示抗轉移劑開發的新目標。

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作者
Chen, Xiangyu.;
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作者單位

City University of New York.;

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授予單位 City University of New York.;
學科 Biology Molecular.;Biology Cell.;Health Sciences Oncology.
學位 Ph.D.
年度 2010
頁碼 95 p.
總頁數 95
原文格式 PDF
正文語種 eng
中圖分類
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Protein kinase C substrates that drive motility of cancer cells.

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