CBL ELISA Kit

Activated epidermal growth factor receptors (EGFRs) are crucial for inducing metastasis in cancer cells by promoting matrix metalloproteinase (MMP) expression. The present study was designed to investigate the effects of 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG) on MMP expression in epidermal growth factor (EGF)-stimulated breast cancer cells in vitro. EGF stimulation induced internalization of its cognate receptor, EGFR, for stimulus-desensitization. These internalized receptors, complexed with the ubiquitin ligase c-Cbl and EGFR pathway substrate 15 (EPS15) (for degradation), were evaluated by confocal microscopy at 5-90 min time intervals. During intracellular trafficking of EGFRs, EGF-induced signaling cascades were analyzed by examining EGFR and SHC phosphorylation. Modulation of MMP expression was assessed by evaluating the activity of transcription factor AP-1 using a luciferase assay. PLAG accelerated the assembly of EGFRs with c-Cbl and EPS15 and promoted receptor degradation. This faster intracellular EGFR degradation reduced AP-1-mediated MMP expression. PLAG stimulation upregulated thioredoxin-interacting protein (TXNIP) expression, and this mediated the accelerated receptor internalization. This PLAG-induced increase in EGFR trafficking was blocked in TXNIP-silenced cells. By downregulating MMP expression, PLAG effectively attenuated EGF-induced mobility and invasiveness in these cancer cells. These data suggest that PLAG may be a potential therapeutic agent for blocking metastasis.

Calcineurin B-like protein interacting protein kinase 6 (CIPK6) is recruited to the tonoplast by Calcineurin B-like protein 2 (CBL2) and interacts with the tonoplast-localized sugar transporter TST2 to promote sugar accumulation, in particular glucose, in cotton. Calcineurin B-like protein (CBL) and CBL-interacting protein kinase (CIPK)-mediated calcium signaling has been widely reported to function in plant development and various stress responses, particularly in ion homeostasis. Sugars are the most important primary metabolites, and thus sugar homeostasis requires precise regulation. Here, we describe a CBL2-CIPK6-Tonoplast-Localized Sugar Transporter2 (TST2) molecular module in cotton (Gossypium hirsutum) that regulates plant sugar homeostasis, in particular Glc homeostasis. GhCIPK6 is recruited to the tonoplast by GhCBL2 and interacts with the tonoplast-localized sugar transporter GhTST2. Overexpression of either GhCBL2, GhCIPK6, or GhTST2 was sufficient to promote sugar accumulation in transgenic cotton, whereas RNAi-mediated knockdown of GhCIPK6 expression or CRISPR-Cas9-mediated knockout of GhTST2 resulted in significantly decreased Glc content. Moreover, mutation of GhCBL2 or GhTST2 in GhCIPK6-overexpressing cotton reinstated sugar contents comparable to wild-type plants. Heterologous expression of GhCIPK6 in Arabidopsis (Arabidopsis thaliana) also promoted Glc accumulation, whereas mutation of AtTST1/2 in GhCIPK6-overexpressing Arabidopsis similarly reinstated wild-type sugar contents, thus indicating conservation of CBL2-CIPK6-TST2-mediated sugar homeostasis among different plant species. Our characterization of the molecular players behind plant sugar homeostasis may be exploited to improve sugar contents and abiotic stress resistance in plants.