Department of Pharmaceutical Sciences, Southern Illinois University Edwardsville, Edwardsville, Illinois, United States of America.
Department of Biomedical Engineering, Saint Louis University, Saint Louis, Missouri, United States of America.
PLoS One. 2017 Dec 14;12(12):e0189589. doi: 10.1371/journal.pone.0189589. eCollection 2017.
IQGAP1 interacts with a number of binding partners through a calponin homology domain (CHD), a WW motif, IQ repeats, a Ras GAP-related domain (GRD), and a conserved C-terminal (CT) domain. Among various biological and cellular functions, IQGAP1 is known to play a role in actin cytoskeleton dynamics during membrane ruffling and lamellipodium protrusion. In addition, phosphorylation near the CT domain is thought to control IQGAP1 activity through regulation of intramolecular interaction. In a previous study, we discovered that IQGAP1 preferentially localizes to retracting areas in B16F10 mouse melanoma cells, not areas of membrane ruffling and lamellipodium protrusion. Nothing is known of the domains needed for retraction localization and very little is known of IQGAP1 function in the actin cytoskeleton of melanoma cells. Thus, we examined localization of IQGAP1 mutants to retracting areas, and characterized knock down phenotypes on tissue culture plastic and physiologic-stiffness hydrogels. Localization of IQGAP1 mutants (S1441E/S1443D, S1441A/S1443A, ΔCHD, ΔGRD or ΔCT) to retracting and protruding cell edges were measured. In retracting areas there was a decrease in S1441A/S1443A, ΔGRD and ΔCT localization, a minor decrease in ΔCHD localization, and normal localization of the S1441E/S1443D mutant. In areas of cell protrusion just behind the lamellipodium leading edge, we surprisingly observed both ΔGRD and ΔCT localization, and increased number of microtubules. IQGAP1 knock down caused loss of cell polarity on laminin-coated glass, decreased proliferation on tissue culture polystyrene, and abnormal spheroid growth on laminin-coated hydrogels. We propose that the GRD and CT domains regulate IQGAP1 localization to retracting actin networks to promote a tumorigenic role in melanoma cells.
IQGAP1 通过一个钙调蛋白同源结构域（CHD）、一个 WW 基序、IQ 重复序列、一个 Ras GAP 相关结构域（GRD）和一个保守的 C 端（CT）结构域与许多结合伴侣相互作用。在各种生物学和细胞功能中，IQGAP1 已知在细胞膜皱襞和片状伪足伸出过程中发挥肌动蛋白细胞骨架动力学的作用。此外，CT 结构域附近的磷酸化被认为通过调节分子内相互作用来控制 IQGAP1 的活性。在之前的研究中，我们发现 IQGAP1 优先定位于 B16F10 小鼠黑色素瘤细胞的回缩区域，而不是细胞膜皱襞和片状伪足伸出的区域。对于回缩定位所需的结构域知之甚少，对于 IQGAP1 在黑色素瘤细胞肌动蛋白细胞骨架中的功能知之甚少。因此，我们检查了 IQGAP1 突变体向回缩区域的定位，并在组织培养塑料和生理硬度水凝胶上对敲低表型进行了表征。测量 IQGAP1 突变体（S1441E/S1443D、S1441A/S1443A、ΔCHD、ΔGRD 或 ΔCT）向回缩和伸出细胞边缘的定位。在回缩区域，S1441A/S1443A、ΔGRD 和 ΔCT 的定位减少，ΔCHD 的定位略有减少，而 S1441E/S1443D 突变体的定位正常。在片状伪足前缘后面的细胞伸出区域，我们令人惊讶地观察到 ΔGRD 和 ΔCT 的定位，以及微管数量的增加。IQGAP1 敲低导致在层粘连蛋白包被玻璃上失去细胞极性，在组织培养聚苯乙烯上增殖减少，以及在层粘连蛋白包被水凝胶上异常的球体生长。我们提出，GRD 和 CT 结构域调节 IQGAP1 向回缩肌动蛋白网络的定位，以促进黑色素瘤细胞的致瘤作用。
