Department of Pharmacology - Head and Neck Surgery, School of Medicine, University of Pennsylvania, 421 Curie Boulevard, BRB II/III, Philadelphia, PA 19104, USA.
Endocr Relat Cancer. 2012 Jul 22;19(4):575-88. doi: 10.1530/ERC-12-0086. Print 2012 Aug.
The functional significance of decreased RAP1GAP protein expression in human tumors is unclear. To identify targets of RAP1GAP downregulation in the thyroid gland, RAP1 and RAP2 protein expression in human thyroid cells and in primary thyroid tumors were analyzed. RAP1GAP and RAP2 were co-expressed in normal thyroid follicular cells. Intriguingly, RAP1 was not detected in normal thyroid cells, although it was detected in papillary thyroid carcinomas, which also expressed RAP2. Both RAP proteins were detected at the membrane in papillary thyroid tumors, suggesting that they are activated when RAP1GAP is downregulated. To explore the functional significance of RAP1GAP depletion, RAP1GAP was transiently expressed at the lowest level that is sufficient to block endogenous RAP2 activity in papillary and anaplastic thyroid carcinoma cell lines. RAP1GAP impaired the ability of cells to spread and migrate on collagen. Although RAP1GAP had no effect on protein tyrosine phosphorylation in growing cells, RAP1GAP impaired phosphorylation of focal adhesion kinase and paxillin at sites phosphorylated by SRC in cells acutely plated on collagen. SRC activity was increased in suspended cells, where it was inhibited by RAP1GAP. Inhibition of SRC kinase activity impaired cell spreading and motility. These findings identify SRC as a target of RAP1GAP depletion and suggest that the downregulation of RAP1GAP in thyroid tumors enhances SRC-dependent signals that regulate cellular architecture and motility.
RAP1GAP 蛋白表达降低在人类肿瘤中的功能意义尚不清楚。为了鉴定 RAP1GAP 在甲状腺下调的靶标，分析了人甲状腺细胞和原发性甲状腺肿瘤中的 RAP1 和 RAP2 蛋白表达。RAP1GAP 和 RAP2 在正常甲状腺滤泡细胞中共同表达。有趣的是，尽管在乳头状甲状腺癌中检测到 RAP1，但在正常甲状腺细胞中未检测到 RAP1，而 RAP2 在这些肿瘤中也有表达。两种 RAP 蛋白都在乳头状甲状腺肿瘤的膜上被检测到，表明当 RAP1GAP 下调时，它们被激活。为了探索 RAP1GAP 耗竭的功能意义，在乳头状和间变性甲状腺癌细胞系中，以足以阻断内源性 RAP2 活性的最低水平瞬时表达 RAP1GAP。RAP1GAP 损害了细胞在胶原蛋白上扩散和迁移的能力。尽管 RAP1GAP 对生长细胞中的蛋白质酪氨酸磷酸化没有影响，但 RAP1GAP 损害了急性铺在胶原蛋白上的细胞中 SRC 磷酸化的粘着斑激酶和桩蛋白的磷酸化。悬浮细胞中 SRC 活性增加，而 RAP1GAP 抑制了其活性。SRC 激酶活性的抑制损害了细胞的扩展和运动。这些发现确定 SRC 为 RAP1GAP 耗竭的靶标，并表明甲状腺肿瘤中 RAP1GAP 的下调增强了调节细胞结构和运动的 SRC 依赖性信号。
