Calreticulin affects cell adhesiveness through differential phosphorylation of insulin receptor substrate-1

Abstract

Cellular adhesion to the underlying substratum is regulated through numerous signaling pathways. It has been suggested that insulin receptor substrate 1 (IRS-1) is involved in some of these pathways, via association with and activation of transmembrane integrins. Calreticulin, as an important endoplasmic reticulum-resident, calcium-binding protein with a chaperone function, plays an obvious role in proteomic expression. Our previous work showed that calreticulin mediates cell adhesion not only by affecting protein expression but also by affecting the state of regulatory protein phosphorylation, such as that of c-src. Here, we demonstrate that calreticulin affects the abundance of IRS-1 such that the absence of calreticulin is paralleled by a decrease in IRS-1 levels and the unregulated overexpression of calreticulin is accompanied by an increase in IRS-1 levels. These changes in the abundance of calreticulin and IRS-1 are accompanied by changes in cell-substratum adhesiveness and phosphorylation, such that increases in the expression of calreticulin and IRS-1 are paralleled by an increase in focal contact-based cellsubstratum adhesiveness, and a decrease in the expression of these proteins brings about a decrease in cell-substratum adhesiveness. Wild type and calreticulin-null mouse embryonic fibroblasts (MEFs) were cultured and the IRS-1 isoform profile was assessed. Differences in morphology and motility were also quantified. While no substantial differences in the speed of locomotion were found, the directionality of cell movement was greatly promoted by the presence of calreticulin. Calreticulin expression was also found to have a dramatic effect on the phosphorylation state of serine 636 of IRS-1, such that phosphorylation of IRS-1 on serine 636 increased radically in the absence of calreticulin. Most importantly, treatment of cells with the RhoA/ROCK inhibitor, Y-27632, which among its many effects also inhibited serine 636 phosphorylation of IRS-1, had profound effects on cell-substratum adhesion, in that it suppressed focal contacts, induced extensive close contacts, and increased the strength of adhesion. The latter effect, while counterintuitive, can be explained by the close contacts comprising labile bonds but in large numbers. In addition, the lability of bonds in close contacts would permit fast locomotion. An interesting and novel finding is that Y-27632 treatment of MEFs releases them from contact inhibition of locomotion, as evidenced by the invasion of a cell's underside by the thin lamellae and filopodia of a cell in close apposition.

Calreticulin nuclear translocalization alleviates CaM/CaMKII/CREB signaling pathway to enhance chemosensitivity in HDAC inhibitor-resistant hepatocellular carcinoma cells

“…TG is a specific inhibitor of the sarcoplasmic/endoplasmic reticulum Ca 2+ -ATPase (SERCA) that causes ER stress through depletion of Ca 2+ store in the ER [63][64][65][66]. CRT acts as a chaperone and affects Ca 2+ homeostasis [67], which influences gene expression and cell adhesion [68]. CRT activated the Wnt and CaM/CaMK II pathways by regulating Ser/Thr phosphatase in regulating cellular adhesion, growth, migration, and differentiation [69][70][71].…”

Mapping the Proximity Interaction Network of STIM1 Reveals New Mechanisms of Cytoskeletal Regulation

“…It is clear that the influence of STIM1 expression and activity on actin-regulatory molecules is a cell-type specific function. To carry out their functions, fibroblasts must migrate within the interstitial space, this requires locally restricted F-actin degradation to facilitate locomotion [37,38]. In contrast, in myocytes focal adhesions are less dynamic, and finely tuned Ca 2+ signaling is essential for regulating important cell function and remodeling other that migration [39].…”