CD44 induced enhancement of phosphatase activity and calcium influx: Modifications of EGR-1 expression and cell proliferation

• CD44 expression reduced Akt phosphorylation and EGR-1 expression.
• CD44 expressing E6.1 Jurkat cells showed increased intracellular Ca2+ concentrations as compared with vector controls.
• Akt phosphorylation and EGR-1 expression were Ca2+ dependent.
• Enhanced phosphatase activity in the CD44 expressing E6.1 Jurkat cells increased the dephosphorylation of Akt; at the same time the increased intracellular Ca2+ concentration ensured that the phosphorylation of Akt remains intact albeit at lower concentrations as compared with the vector control.
• Reduced Akt phosphorylation resulted in lowered expression of EGR-1 and reduced the cell proliferation rate.

The purpose of this study was to investigate how CD44 impaired Akt phosphorylation, EGR-1 expression and cell proliferation. E6.1 Jurkat cells, which lack endogenous CD44 expression, were engineered to express CD44. Previously we showed that Akt is hypophosphorylated, EGR-1 expression is reduced and proliferation is impaired in CD44 expressing E6.1 Jurkat cells. The cell cycle was studied using flow cytometry and the role of calcium (Ca2+) in Akt phosphorylation and EGR-1 expression was investigated using Western blotting. Phosphatase activity was assessed using a commercially available kit. CD44 expressing cells showed disruption at the G1 to S transition. Chelation of Ca2+ from the culture media impaired Akt phosphorylation and EGR-1 expression in both CD44 expressing cells and the open vector control. Moreover, Ni2+ disrupted cell proliferation in both cell types suggesting Ca2+ import through calcium release activated calcium channels (CRAC). Staining of cells with fura-2 AM showed significantly higher Ca2+ in CD44 expressing cells as compared with the vehicle control. Finally, non-calcium mediated phosphatase activity was significantly greater in CD44 expressing cells. We propose that the enhanced phosphatase activity in the CD44 cells increased the dephosphorylation rate of Akt; at the same time, the increased intracellular concentration of Ca2+ in the CD44 cells ensured that the phosphorylation of Akt remains intact albeit at lower concentrations as compared with the vector control. Reduced Akt phosphorylation resulted in lowered expression of EGR-1 and hence, reduced the cell proliferation rate.