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Protein kinase C substrates that drive motility of cancer cells.

机译：驱动癌细胞运动的蛋白激酶C底物。

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摘要

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.
机译：作为肿瘤促进佛波酯的细胞内受体，蛋白激酶C（PKC）在功能上与致癌和转移有关。因此，至关重要的是鉴定PKC的底物，以了解这些底物蛋白参与癌症相关表型（如运动行为）的机制。待描述的工作包括两个项目：（1）有助于人类乳腺癌细胞运动表型的新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。为了鉴定直接底物，使用化学遗传学方法对PKC-α，δ和＆zgr的ATP结合位点进行工程改造。使它们能够结合非天然的ATP类似物。因此，磷酸化仅归因于突变酶，而磷酸化的蛋白带是潜在的底物。在人乳腺细胞（MCF-10A）中表达后，对与高亲和力蛋白质底物结合的突变酶进行了免疫共沉淀。加入ATP类似物后，产生了许多磷酸化蛋白，随后通过质谱分析。 PKC-α和δ可追踪的激酶具有非常相似的磷酸化模式，而PKC-＆zgr的分布则为磷酸化。是与众不同的。 PKC-alpha和PKC-delta的几种可能的蛋白质底物都已被发现，包括与小GTP结合蛋白结合的蛋白（Rho Kinase-1，Cdc42ep4，Rho / cdc42 / Rac活化蛋白-1）以及以前已被证明是PKC底物的蛋白质（IQGAP，VASP）。；在第二个项目中，研究了MARCKS在小鼠黑色素瘤细胞运动中的作用。作为已知将肌动蛋白束交联到质膜的蛋白质，这种PKC底物与细胞迁移和粘附有关。通过PKC磷酸化后，MARCKS释放到细胞质中，从而促进肌动蛋白重排。转移性弱的B16 F1细胞不表达可检测的磷酸化MARCKS。但是，被工程化以表达MARCKS假磷酸化突变体的F1细胞表现出提高的运动能力。当F1细胞用蛋白磷酸酶的抑制剂冈田酸（OA）处理时，可观察到磷酸MARCKS和运动性均增加。当用shRNA试剂预处理细胞以降低MARCKS的表达，或表达抗磷酸化的MARCKS突变体时，可以基本上消除OA诱导的运动。此外，观察到由于缺乏肉豆蔻酰基而仅具有胞质性的抗磷酸化的MARCKS突变体抑制了OA处理的F1细胞的运动以及高度转移性黑素瘤细胞（小鼠F10和人A375细胞）的运动。这些发现暗示MARCKS通过涉及其磷酸化的效应子结构域的细胞质相互作用促进运动。结论是，磷酸-MARCKS在细胞质中具有以前未记载的作用，可促进许多不同癌细胞的运动行为以及可能的转移潜能。总体而言，这项工作描述了在癌细胞中传递运动信号的PKC底物，并暗示抗转移剂开发的新目标。

著录项

  • 作者

    Chen, Xiangyu.;

  • 作者单位

    City University of New York.;

  • 授予单位 City University of New York.;
  • 学科 Biology Molecular.;Biology Cell.;Health Sciences Oncology.
  • 学位 Ph.D.
  • 年度 2010
  • 页码 95 p.
  • 总页数 95
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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