Differential regulation of class IA phosphoinositide 3-kinase catalytic subunits p110 alpha and beta by protease-activated receptor 2 and beta-arrestins

Abstract

PAR-2 (protease-activated receptor 2) is a GPCR (G-protein-coupled receptor) that can elicit both G-protein-dependent and -independent signals. We have shown previously that PAR-2 simultaneously promotes Galphaq/Ca2+-dependent activation and beta-arrestin-1-dependent inhibition of class IA PI3K (phosphoinositide 3-kinase), and we sought to characterize further the role of beta-arrestins in the regulation of PI3K activity. Whereas the ability of beta-arrestin-1 to inhibit p110alpha (PI3K catalytic subunit alpha) has been demonstrated, the role of beta-arrestin-2 in PI3K regulation and possible differences in the regulation of the two catalytic subunits (p110alpha and p110beta) associated with p85alpha (PI3K regulatory subunit) have not been examined. In the present study we have demonstrated that: (i) PAR-2 increases p110alpha- and p110beta-associated lipid kinase activities, and both p110alpha and p110beta are inhibited by over-expression of either beta-arrestin-1 or -2; (ii) both beta-arrestin-1 and -2 directly inhibit the p110alpha catalytic subunit in vitro, whereas only beta-arrestin-2 directly inhibited p110beta; (iii) examination of upstream pathways revealed that PAR-2-induced PI3K activity required the small GTPase Cdc (cell-division cycle)42, but not tyrosine phosphorylation of p85; and (iv) beta-arrestins inhibit PAR-2-induced Cdc42 activation. Taken together, these results indicated that beta-arrestins could inhibit PAR-2-stimulated PI3K activity, both directly and through interference with upstream pathways, and that the two beta-arrestins differ in their ability to inhibit the p110alpha and p110beta catalytic subunits. These results are particularly important in light of the growing interest in PAR-2 as a pharmacological target, as commonly used biochemical assays that monitor G-protein coupling would not screen for beta-arrestin-dependent signalling events.

Figure 1. Expression of p110α and p110β subunits in NIH 3T3 cells

(A) Specificity of antibodies in NIH 3T3 cell lysates. Lysates were immunoprecipitated (IP) with anti-IgG and anti-p110β antibodies (left-hand side panel) or anti-IgG and anti-p110α antibodies (right-hand side panel), followed by Western blotting with antibodies against p110α, p110β and p85. No p110α was co-immunoprecipitated with anti-p110β antibody and vice versa. (B) Protein expression of p110α and p110β in NIH 3T3 whole-cell lysates. Representative Western blots probed with anti-p110α and anti-actin antibodies (top panel) or p110β and actin (bottom panel). (C) mRNA expression of p110α and p110β in NIH 3T3 cells. cDNA was synthesized from total RNA isolated from NIH 3T3 cells, and used as a template for PCR. A representative agarose gel of PCR products using specific primers directed against p110α, p110β and GAPDH (internal control) is shown.

Figure 2. PAR-2 activates both p110α and p110β subunits of the class IA PI3K kinase

NIH 3T3 cells were treated with or without 100 nM 2fAP (PAR-2 activating peptide) or 100 nM IGF-1 for 5 min, lysed and immunoprecipitated (IP) with antibodies against p110α or p110β. Immune complexes were incubated with PtdIns in the presence of [γ-³²P]ATP and phospholipids were separated by TLC to determine PI3K activity. (A) Western blot with anti-IgG and anti-p85 antibodies to demonstrate equal immunoprecipitation levels. (B) Representative autoradiograph of phospholipids separated by TLC. Migration rates of PtdIns product (PIP) were compared with rates of stained standards, and the migration position is indicated by an arrow. A full TLC plate with both origin and phospholipids is shown. (C) Histogram depicting baseline PI3K activity in p110α and p110β immunoprecipitates as normalized phosphate incorporation into PtdIns (left-hand side panel), and fold increase in PI3K activity after treatment with 2fAP or IGF-1 as a fraction baseline (right-hand side panel). (D) PI3K assay was performed as described above using PtdIns(4,5)P₂ as a substrate. Incorporation of phosphate in all PI3K assays was determined by phosphoimage analysis. Fold change was determined by the ratio of band density in treated samples compared with untreated samples. Arbitrary units refer to raw band density, as determined by phosphoimage analysis. All densities were normalized to levels of p85 in p110α or p110β immunoprecipitates.

Figure 3. PAR-2 inhibits p110α and p110β activity through both β-arrestin-1 and β-arrestin-2

NIH 3T3 cells transfected with FLAG-tagged β-arrestin-1 (Flag-βarr1), β-arrestin-2 (Flag-βarr2) or empty vector as a control (Cnt) were treated with 100 nM 2fAP for 0 or 5 min, and PI3K activity was measured from samples immunoprecipitated (IP) using anti-p110α (A) and anti-p110β (B) antibodies as performed in Figure 2. A representative autoradiograph of phospholipids separated by TLC is shown, where PIP is the PtdIns product and a histogram showing PI3K activity in response to 2fAP as fraction baseline. (C) Representative Western blot showing anti-FLAG antibody immunoreactivity in cells transfected with empty vector as a control (cnt) or either FLAG–β-arrestin-1 (Flag-βarr1) or FLAG–β-arrestin-2 (Flag-βarr2).

Figure 4. Effect of β-arrestins on the catalytic activity of p110α and p110β in vitro

(A,B) Increasing concentrations (nM) of GST–β-arrestin-1 (β1GST), GST–β-arrestin-2 (β2GST) or GST alone (negative control) were added to reaction mixtures containing [γ-³²P]ATP, purified PtdIns and either recombinant p85/p110α (A) or recombinant p85/p110β (B). Upper panels show representative autoradiographs of phospholipids separated by TLC. Lower panels show semi-log plots of PI3K activity (defined as the fraction of control) as a function of the GST fusion protein concentration (in M). The control activity is that observed in the absence of added GST fusion protein. (C,D) Increasing amounts of anti-FLAG antibody immunoprecipitates from NIH 3T3 cells transfected with FLAG–β-arrestin-1 (β1-Flag), FLAG–β-arrestin-2 (β2-Flag) or empty vector as a control (CT) were added to in vitro recombinant p85/p110α (C) and recombinant p85/p110β (D) and kinase activity was measured as described above. The amount of anti-FLAG–agarose added (20 and 40 μl) corresponded to approx. 500 and 1000 ng of β-arrestin-1 and approx. 50 and 300 ng of β-arrestin-2. Upper panels show representative autoradiographs of phospholipids separated by TLC. Lower panels show plots of PI3K activity as a function of β-arrestin added (in ng). PI3K activity is defined as the fraction of control (activity observed in the presence of an equal volume of anti-FLAG immunoprecipitates from untransfected cells). (E) Representative Coomassie-stained gels of increasing amounts of β-arrestin-2–GST (β-arr-2GST) and GST (left-hand side panel) and β-arrestin-1–GST (β1GST) (right-hand side panel). (F) Representative Coomassie-stained gel of increasing amounts of FLAG–β-arrestin-1 (β1-Flag) and FLAG–β-arrestin-2 (β2-flag).

Figure 5. PAR-2-stimulated PI3K activity requires Cdc42 but not p85 tyrosine phosphorylation

(A) NIH 3T3 cells were treated with or without 100 nM 2fAP for 5 min and immunoprecipitated (IP) with anti-IgG or anti-p85 antibodies, followed by Western blotting with anti-phospho-tyrosine (upper left-hand side panel). Blots were then reprobed with anti-p85 antibody (upper right-hand side panel), then stripped and the region corresponding to 100–200 kDa was reprobed with anti-p110 antibody, which recognizes both p110α and p110β (lower panel). (B–D) NIH 3T3 cells were transfected with EGFP (enhanced GFP) N1 control vector (CNT), DNRHO, DNCDC or DNRAC, treated with or without 2fAP, and PI3K activity in anti-p85 antibody immunoprecipitates was determined as described above. (B) Representative autoradiograph of phospholipids separated by TLC. (C) Histogram depicting PAR-2-induced PI3K activity (as fraction of baseline) under each transfected condition. (D) Representative Western blot of GTPase over-expression (upper panel: anti-GFP antibody; lower panel: anti-HA-7 antibody).

Figure 6. β-arrestin-1 and β-arrestin-2 inhibit PAR-2-stimulated Cdc42 activity

(A–B) NIH 3T3 cells were treated with or without 2fAP or serum (ser) as a positive control, and lysates were incubated with either PAK3 CRIB–GST or Rhotekin–GST. Total lysates and bound proteins were analysed by SDS/PAGE followed by Western blotting with anti-Cdc42, anti-Rac-1 or anti-RhoA antibodies. (A) Representative Western blots, where the left-hand side lanes contain total lysates and right-hand side lanes contain bound proteins. (B) Histogram depicting PAR-2 and serum-induced changes in CRIB domain binding (as a fraction of baseline). (C–D) MEFwt, MEF β-arrestin-1, β-arrestin-2-DKO, MEF β-arrestin-1, β-arrestin-2-DKO with β-arrestin-1 (DKO+βarr1) and MEF β-arrestin-1, β-arrestin-2-DKO with β-arrestin-2 (DKO+βarr2) cells were treated with 100 nM 2fAP for 0 or 5 min and activation of Cdc42 was determined by association with GST–PAK3 CRIB domain. (C) Representative Western blot of bound proteins (B) and total lysates (TL) blotted with anti-Cdc42 antibody. (D) Histogram depicting PAR-2-stimulated changes in Cdc42/CRIB domain binding (as a fraction of baseline) in each cell line. Unt, untreated.

Figure 7. Model of PI3K regulation by PAR-2.

(A) Gαq compared with β-arrestin-dependent pathways. On activation, PAR-2 promotes Gαq coupling and subsequent mobilization of intracellular Ca²⁺ and activation of PKC (i). Ca²⁺ and PKC can promote activation of PYK2 and Src [1,11], association of PYK2 with p85 [7] and possibly phosphorylation of p110 (shown in Figure 5) (ii). PAR-2 also promotes activation of Cdc42 by an unknown mechanism (shown in Figures 5 and 6), perhaps through Gαq/Ca²⁺-dependent mechanisms or through coupling to a different G-protein (iii). Activation of Cdc42 and Src leads to activation of p110α and p110β (p110*) (iv). β-Arrestin signalling through PAR-2 opposes the Gαq pathway through multiple mechanisms. First, PAR-2 promotes recruitment of both β-arrestins and p85/p110 to the receptor [1,7,17] (v), which can lead to inhibition of p110 catalytic activity (vi). β-Arrestins also inhibit PAR-2-stimulated Cdc42 activation (shown in Figure 6) (vii) and promote receptor uncoupling from Gαq [17] (viii), both of which would inhibit PAR-2-induced PI3K activity. (B) Model of IGF-1-stimulated PI3K activity (for comparison with PAR-2). IGF-1 has been shown to promote tyrosine phosphorylation of p85 SH2 domains, and its recruitment to phospho-tyrosines on IRS-1 or the IRS-1 receptor itself [18,19] (i). β-Arrestins were shown to facilitate IRS-1-induced PI3K activity, possibly through its ability to bind both the IGF-1R and SH3 domain-containing proteins [8,24] (ii). This may indirectly recruit the p85/p110 complex to the receptor. (C) PAR-2 can promote β-arrestin-dependent inhibition of both p110 subunits. Both β-arrestin-1 and β-arrestin-2 can directly inhibit p110α in vitro (i), but can only do so in vivo on PAR-2 activation (ii). Only β-arrestin-2 can directly inhibit p110β in vitro (iii), however, on activation of PAR-2 in vivo β-arrestin-1 becomes inhibitory. This may reflect an association with a third factor (“factor X”) in cells (iv) or post-translational modification of the receptor, e.g. phosphorylation (v).