The phosphatidylinositol (PI)-5-phosphate 4-kinase type II enzyme controls insulin signaling by regulating PI-3,4,5-trisphosphate degradation

Abstract

Phosphatidylinositol-5-phosphate (PI-5-P) is a newly identified phosphoinositide with characteristics of a signaling lipid but no known cellular function. PI-5-P levels are controlled by the type II PI-5-P 4-kinases (PIP4K IIs), a family of kinases that converts PI-5-P into phosphatidylinositol-4,5-bisphosphate (PI-4,5-P2). The PI-5-P pathway is an alternative route for PI-4,5-P2 synthesis as the bulk of this lipid is generated by the canonical pathway in which phosphatidylinositol-4-phosphate (PI-4-P) is the intermediate. Here we examined the effect of activation of the PI-5-P pathway on phosphoinositide 3-kinase (PI3K) signaling by expressing PIP4K II beta in cells that lack this enzyme. Although PIP4K II generates PI-4,5-P2, a substrate for PI3K, expression of this enzyme reduced rather than increased phosphatidylinositol-3,4,5-trisphosphate (PI-3,4,5-P3) levels in cells stimulated with insulin or cells expressing activated PI3K. This reduction in PI-3,4,5-P3 levels resulted in decreased activation of the downstream protein kinase, Akt/PKB. Consistent with these results, expression of IpgD, a bacterial phosphatase that converts PI-4,5-P2 to PI-5-P, resulted in Akt activation, and this effect was partially reversed by PIP4K II beta. PIP4K II beta expression did not impair insulin-dependent association of PI3K with insulin receptor substrate 1 (IRS1) but abbreviated Akt activation, indicating that PIP4K II regulates PI-3,4,5-P3 degradation rather than synthesis. These data support a model in which the PI-5-P pathway controls insulin signaling that leads to Akt activation by regulating a PI-3,4,5-P3 phosphatase.

Fig. 1.

The PI-5-P pathway for PI-4,5-P₂ synthesis regulates the insulin-induced Akt phosphorylation. CHO-IR cells were transfected with HA-Akt and with the genes indicated above each panel. Cells were serum-starved for 24 h and stimulated (as indicated) with insulin (i, 10 nM) or serum (s, 20%) for 10 min. Phosphorylation of the transfected HA-Akt was assayed by using phospho-specific antibodies against pT308 or pS473 of Akt. Also shown are the Western blots of the total cell lysates with antibodies against the transfected proteins or against pTyr. Results are representative of three or more experiments. IP, immunoprecipitation.

Fig. 2.

PIP4K IIβ decreases PI-3,4,5-P₃ levels downstream of PI3K activation. CHO-IR cells were transfected with PIP4K IIβ or empty vector (V) as indicated and serum-starved for 24 h. (A) Cells were labeled for 4 h with [³²P]orthophosphate and stimulated with insulin (10 nM) for 5 min. The deacylated lipids were analyzed by HPLC, quantified, and normalized as described. The graph represents the relative levels of PI-3,4,5-P₃ (black) or PI-3,4-P₂ (gray) when compared with the PI-3,4,5-P₃ lvels in cells transfected with empty vector. (B) Cell lysates were immunoprecipitated (IP) with anti-pTyr antibody and blotted with anti-pTyr antibody or anti-p85 antibody or assayed for PI3K activity. (C) CHO-IR cells were transfected with GFP-p85-Flag and with PIP4K IIβ or empty vector (V). Protein lysates were immunoprecipitated with anti-flag antibody and blotted with anti-pTyr or anti-GFP antibodies. Total cell lysates were blotted with anti-PIP4K IIβ antibody. Results are representative of two or more experiments.

Fig. 3.

PIP4K IIβ decreases PI-3,4,5-P₃, but not PI-3,4-P₂ levels. (A) CHO-IR cells were transfected with p110caax and with PIP4K IIβ, Ship2, or empty vector (V) as indicated. Cells were serum-starved and labeled with [³H]inositol for 24 h. The deacylated lipids were analyzed by HPLC, quantified, and normalized as described. The graphs represent the relative levels of PI-3,4,5-P₃ (black) or PI-3,4-P₂ (gray) when compared with the PI-3,4,5-P₃ levels in cells transfected with empty vector. (B and C) The ratio of PI-3,4,5-P₃ and PI-3,4-P₂ in cells expressing p110caax (B) or cells stimulated with insulin (C). The gray bars represent the percentage of PI-3,4-P₂, and the black bars represent the percentage of PI-3,4,5-P₃ over the sum of PI-3,4-P₂ and PI-3,4,5-P₃. The error bars represent the range from two experiments.

Fig. 4.

PIP4K IIβ causes premature termination of the insulin signal. CHO-IR cells were transfected with HA-Akt and with PIP4K IIβ or vector control. Cells were serum-starved for 24 h and stimulated with insulin. Phosphorylation of the transfected HA-Akt was assayed by using phospho-specific antibodies against pT308 of Akt. (A) Cells were stimulated with 1 nM insulin for 10, 15, 20, or 25 min as indicated. Total cell lysates were blotted with antibodies against PIP4K IIβ or endogenous Ship2. The bar graph shows the levels of phospho-Akt in PIP4K IIβ-expressing cells as a fraction of the levels of phospho-Akt in vector-transfected cells. The quantification was performed by using two independent experiments. (B) Cells were stimulated with 10 nM insulin for 5 min and treated with DMSO or wortmannin (Wortm., 100 nM) for 2.5–10 min. The bar graph is a plot of the results obtained from two independent experiments. The data were normalized by calculating the fraction of Akt activation in wortmannin-treated cells relative to DMSO-treated cells. IP, immunoprecipitation.

Fig. 5.

Inactivation of PIP4K II and IpgD impairs their ability to regulate Akt phosphorylation. CHO-IR cells were transfected with HA-Akt and with wild-type or D278A PIP4K IIβ (A) or wild-type or C438S GFP-IpgD (B). Cells were serum-starved for 24 h and stimulated with insulin (10 nM) for 10 min (A) or left untreated (B). Lysates were prepared, immunoprecipitated (IP) with anti-HA, and assayed for phosphorylated Akt by using anti-Akt pT308 antibody. The result shown is representative of two or more separate experiments.

Fig. 6.

Schematic diagram showing our current model for the role of PIP4K II in PI-3,4,5-P₃ regulation. PIP4K II expression in cells stimulates PI-3,4,5-P₃ degradation. Our results are consistent with PIP4K II activating a PI-3,4,5-P₃-specific 5-phosphatase. However, we have not eliminated the possibility that a PI-3,4,5-P₃-specific 3-phosphatase is also activated by PIP4K II. The thick arrows indicate a pathway, and the thin arrows indicate a biochemical reaction.