Cytoplasmic Domain of proEGF Affects Distribution and Post-Translational Modification of Microtubuli and Increases Microtubule-Associated Proteins 1b and 2 Production in Human Thyroid Carcinoma Cells

Abstract

We studied the distribution of transcripts encoding the cytoplasmic domain of the membrane-anchored precursor epidermal growth factor (proEGFcyt) and a novel cytoplasmic proEGF splice isoform with a deleted exon 23 and an out-offrame fusion of exon 24 (proEGFdel23) in human normal and neoplastic thyroid tissues. In papillary thyroid carcinoma (PTC), coexpression of transcripts encoding for both proEGFcyt and proEGFdel23 correlated with poor differentiation of PTC. To determine potential roles of the cytoplasmic proEGF domain in human thyroid cells, we generated stable transfectants of the human follicular thyroid carcinoma cell line FTC-133 overexpressing the normal cytoplasmic domain proEGFcyt, a truncated proEGFcyt composed of the peptide sequence encoded by exons 22 and 23 (proEGF22.23) and proEGFdel23. The proEGFcyt and proEGF22.23 transfectants displayed significantly reduced proliferation rates, an enlarged cellular phenotype, and alterations in the distribution and post-translational modification of the microtubular system. These transfectants also displayed increased production of microtubule-associated proteins 1b and 2c, which was absent in FTC-133-proEGFdel23 or FTC-133-empty plasmid transfectants. This is the first evidence of an involvement of proEGF cytoplasmic domain in microtubular stability in the human thyroid carcinoma cell line FTC-133 and may suggest a specific role for the cytoplasmic domain of membraneanchored proEGF, particularly exon 23, in thyroid carcinoma. The up-regulation of proEGFdel23 in poorly differentiated PTC and the exclusive detection of both proEGF isoforms in undifferentiated thyroid carcinoma may indicate an involvement of this novel truncated proEGFdel23 cytoplasmic domain during dedifferentiation processes of human thyroid cells. (Cancer Res 2005; 65(4): 1343-51)

The epidermal growth factor receptor ligands at a glance

“…These include, most notably, to mediate the correct trafficking and delivery of TGFA to the cell surface (Fernandez‐Larrea et al, 1999) and to carry basolateral sorting information for TGFA (Dempsey et al, 2003) and AREG (Brown et al, 2001). Recently, the CT of EGF was shown to alter the distribution and post‐translational modification of the microtubular system and to increase the production of microtubule‐associated proteins 1b and 2 in human thyroid carcinoma cells (Pyka et al, 2005). While the main task of the CT may be related to ligand trafficking and delivery to the cell surface, a new, exciting possibility emerged with the identification of the transcriptional repressors promyelocytic leukemia zinc finger (PLZF, Nanba et al, 2003) and B‐cell leukemia 6 (Bcl6, Kinugasa et al, 2007) as binding proteins of HBEGF CT (Fig.…”

The C‐terminal cytoplasmic domain of human proEGF is a negative modulator of body and organ weights in transgenic mice

“…Total RNA isolation, first strand cDNA synthesis, and semiquantitative RT‐PCR were performed as described earlier [2,3]. Primers and PCR conditions were used as described previously [15].…”

“…An Eco RI fragment of human proEGFcyt [3] was cloned downstream of the cytomegalovirus enhancer, chicken β‐actin promoter and rabbit β‐globin splice acceptor, and upstream to the rabbit β‐globin 3′‐flanking region and polyadenylation signal in the expression vector pUC‐CAGGS (Fig. 1 A).…”

“…The membrane-anchored proform of the largest epidermal growth factor (EGF)-like precursor, proEGF, contains the largest cytoplasmic domain (155 aa; proEGFcyt) of all EGFR ligands [1]. Cytoplasmic domains of proEGF-like ligands are biologically active and have multiple cellular functions [2][3][4][5][6][7][8]. Transgenic (tg) mice expressing human full-length proEGF under the control of a beta-actin promoter showed stunted growth, abnormalities of osteoblasts, focal liver necrosis, and infertility [9,10].…”

Sequential and γ-secretase-dependent processing of the betacellulin precursor generates a palmitoylated intracellular-domain fragment that inhibits cell growth

“…For example, the cytoplasmic domains of several ErbB-ligand precursors regulate polarized trafficking and cell-surface expression (Brown et al, 2001;Dempsey and Coffey, 1994;Dempsey et al, 2003;Dempsey et al, 1997;Dong and Wiley, 2000), whereas the membrane-anchoring domain of the HB-EGF precursor (proHB-EGF) can confer juxtacrine activity (Dong et al, 2005). Moreover, accessory proteins, such as CD9, that directly interact with the cytoplasmic domains of specific ErbB-ligand precursors can also modify ligand function (Franklin et al, 2005;Glogowska et al, 2008;Imhof et al, 2008;Klonisch et al, 2009;Pyka et al, 2005;Shi et al, 2000;Shum et al, 1994;Shum et al, 1996). In addition, other post-translational modifications of ErbB-ligand precursors, including phosphorylation and palmitoylation of the cytoplasmic domain, are likely to further modulate these protein interactions and signaling events (Shum et al, 1996;Wang et al, 2006).…”