Bourguignon Lilly Y W, Gilad Eli, Brightman Amy, Diedrich Falko, Singleton Patrick
Department of Medicine, University of California at San Francisco and Endocrine Unit (111N), Veterans Affairs Medical Center, San Francisco, California 94121, USA.
J Biol Chem. 2006 May 19;281(20):14026-40. doi: 10.1074/jbc.M507734200. Epub 2006 Mar 24.
In this study we have examined the interaction of CD44 (a major hyaluronan (HA) receptor) with a RhoA-specific guanine nucleotide exchange factor (leukemia-associated RhoGEF (LARG)) in human head and neck squamous carcinoma cells (HNSCC-HSC-3 cell line). Immunoprecipitation and immunoblot analyses indicate that CD44 and the LARG protein are expressed in HSC-3 cells and that these two proteins are physically associated as a complex. HA-CD44 binding induces LARG-specific RhoA signaling and phospholipase C epsilon (PLC epsilon) activity. In particular, the activation of RhoA-PLC epsilon by HA stimulates inositol 1,4,5-triphosphate production, intracellular Ca2+ mobilization, and the up-regulation of Ca2+/calmodulin-dependent kinase II (CaMKII), leading to phosphorylation of the cytoskeletal protein, filamin. The phosphorylation of filamin reduces its interaction with filamentous actin, promoting tumor cell migration. The CD44-LARG complex also interacts with the EGF receptor (EGFR). Most importantly, the binding of HA to the CD44-LARG-EGFR complex activates the EGFR receptor kinase, which in turn promotes Ras-mediated stimulation of a downstream kinase cascade including the Raf-1 and ERK pathways leading to HNSCC cell growth. Using a recombinant fragment of LARG (the LARG-PDZ domain) and a binding assay, we have determined that the LARG-PDZ domain serves as a direct linker between CD44 and EGFR. Transfection of the HSC-3 cells with LARG-PDZcDNA significantly reduces LARG association with CD44 and EGFR. Overexpression of the LARG-PDZ domain also functions as a dominant-negative mutant (similar to the PLC/Ca2+-calmodulin-dependent kinase II (CaMKII) and EGFR/MAPK inhibitor effects) to block HA/CD44-mediated signaling events (e.g. EGFR kinase activation, Ras/RhoA co-activation, Raf-ERK signaling, PLC epsilon-mediated inositol 1,4,5-triphosphate production, intracellular Ca2+ mobilization, CaMKII activity, filamin phosphorylation, and filamin-actin binding) and to abrogate tumor cell growth/migration. Taken together, our findings suggest that CD44 interaction with LARG and EGFR plays a pivotal role in Rho/Ras co-activation, PLC epsilon-Ca2+ signaling, and Raf/ERK up-regulation required for CaMKII-mediated cytoskeleton function and in head and neck squamous cell carcinoma progression.
在本研究中，我们检测了人头颈鳞状癌细胞（HNSCC-HSC-3细胞系）中CD44（一种主要的透明质酸（HA）受体）与RhoA特异性鸟嘌呤核苷酸交换因子（白血病相关Rho鸟嘌呤核苷酸交换因子（LARG））之间的相互作用。免疫沉淀和免疫印迹分析表明，CD44和LARG蛋白在HSC-3细胞中表达，且这两种蛋白以复合物形式物理结合。HA-CD44结合诱导LARG特异性RhoA信号传导和磷脂酶Cε（PLCε）活性。特别地，HA对RhoA-PLCε的激活刺激了肌醇1,4,5-三磷酸的产生、细胞内Ca2+动员以及Ca2+/钙调蛋白依赖性激酶II（CaMKII）的上调，导致细胞骨架蛋白细丝蛋白的磷酸化。细丝蛋白的磷酸化减少了其与丝状肌动蛋白的相互作用，促进肿瘤细胞迁移。CD44-LARG复合物还与表皮生长因子受体（EGFR）相互作用。最重要的是，HA与CD44-LARG-EGFR复合物的结合激活了EGFR受体激酶，进而促进Ras介导的对包括Raf-1和ERK途径在内的下游激酶级联反应的刺激，导致HNSCC细胞生长。使用LARG的重组片段（LARG-PDZ结构域）和结合试验，我们确定LARG-PDZ结构域作为CD44和EGFR之间的直接连接物。用LARG-PDZ cDNA转染HSC-3细胞可显著降低LARG与CD44和EGFR的结合。LARG-PDZ结构域的过表达还作为一种显性负性突变体（类似于PLC/Ca2+-钙调蛋白依赖性激酶II（CaMKII）和EGFR/MAPK抑制剂的作用）来阻断HA/CD44介导的信号事件（如EGFR激酶激活、Ras/RhoA共激活、Raf-ERK信号传导、PLCε介导的肌醇1,4,5-三磷酸产生、细胞内Ca2+动员、CaMKII活性、细丝蛋白磷酸化以及细丝蛋白-肌动蛋白结合）并消除肿瘤细胞生长/迁移。综上所述，我们的研究结果表明，CD44与LARG和EGFR的相互作用在Rho/Ras共激活、PLCε-Ca2+信号传导以及CaMKII介导的细胞骨架功能和头颈鳞状细胞癌进展所需的Raf/ERK上调中起关键作用。
