The p85beta regulatory subunit of PI3K serves as a substrate for PTEN protein phosphatase activity during insulin mediated signaling

Abstract

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a tumor suppressor gene that is frequently mutated in brain, uterine, and prostate cancers. The protein phosphatase activity is poorly defined. We demonstrate that insulin stimulates phosphorylation of tyrosine and threonine/proline residues on the p85 regulatory subunit of PI3K in Huh-7, and HEK 293 cells. The specificity of PTEN binding and dephosphorylation of PI3K appears to reside on the p85beta subunit. Therefore, the PTEN phosphatase is active against the PI3K p85beta subunit and dephosphorylates a protein involved in insulin signaling where known downstream consequences are increased cell migration, motility, and invasion.

Fig. 1

Insulin stimulation induces phosphorylation of the p85 subunit of PI3K on tyrosine and threonine-proline residues and activates Akt (pAkt). (A) Huh7 cells or (C) HEK 293 T-cells were stimulated with insulin or vehicle (control) for 10 minutes after an overnight serum starvation. Note the phosphorylation of p85 and the activation of Akt in both cell lines. (B) Huh7 cells or (D) HEK 293 T-cells were transfected with a WT PTEN expression plasmid, or an empty vector control and then stimulated with insulin or vehicle. Note that WT PTEN dephosphorylates Tyr and Thr-Pro residues on the p85 subunit of PI3K following insulin stimulation.

Fig. 2

The C124S and G129E mutations in the catalytic site for phosphatase activitypreserve Akt activation following insulin stimulation. Cell lysates were analyzed by immunoblotting using antibodies to phospho-Akt, PTEN, and actin. Note that WT PTEN inhibits phosphorylation of Akt both with and without insulin stimulation, whereas the C124S and G129E mutant constructs do not. (A). The right hand panel represents densiometric scanning of the Western blots presented in Panel A. The experiment was repeated three times with similar results. (C). The PTEN C124S mutant has no protein phosphatase activity for the p85 subunit of PI3K following insulin stimulation. HEK 293 T-cells were transfected with expression plasmids and stimulated with insulin. Note that insulin stimulated p-tyrosine and p-threonine-proline on p85. However, the C124S mutant exhibited no protein phosphatase activity whereas the G129 mutant behaved like WT PTEN and reduced insulin stimulated p-tyrosine and p-threonine proline phosphorylation of p85.

Fig. 3

PTEN dephosphorylates p-tyrosine and p-threonine-proline on the p85β subunit of PI3K. (A). Densiometric scanning of the Western blots are shown in Panel B. (B). Note that PTEN phosphatase is active for the p85β but not the p85α subunit of PI3K.

Fig. 4

The PTEN C124S mutant does not bind to p85 subunit of PI3K. HEK 293 T-cells were transfected with expression plasmids, control, or PTEN 1–274 and 1–336 N-terminal deletion mutants, along with p85 expression plasmid and stimulated with insulin (0.075U/ml). Densiometric scanning and Western blot are presented in Panel A (left and right, respectively). B. Cell lysates were analyzed by immunoblotting for PTEN deletion mutant binding as well as determining the expression levels of different PTEN mutant proteins. Note that the PTEN C124S mutant does not bind to the p85 subunit of PI3K whereas WT PTEN, G129E and 1–336, and 1–273 deletion mutants physically interact with the p85 subunit of PI3K. (C). The PTEN I-273, (I-336, C124S and G129E mutants do not inhibit insulin stimulated pAkt formation compared to WT PTEN.