Targeting PI3Kγ anchoring enhances CFTR membrane localization and modulator efficacy via PKD1
Alessandra Murabito, Marco Mergiotti, Valeria Capurro, Alessia Loffreda, Mingchuan Li, Paola Peretto, Kai Ren, Andrea Raimondi, Carlo Tacchetti, Dario Diviani, Nicoletta Pedemonte, Emilio Hirsch, Alessandra Ghigo
Alessandra Murabito, Marco Mergiotti, Valeria Capurro, Alessia Loffreda, Mingchuan Li, Paola Peretto, Kai Ren, Andrea Raimondi, Carlo Tacchetti, Dario Diviani, Nicoletta Pedemonte, Emilio Hirsch, Alessandra Ghigo
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Research Article Cell biology Pulmonology

Targeting PI3Kγ anchoring enhances CFTR membrane localization and modulator efficacy via PKD1

Abstract

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a cAMP-activated chloride channel, cause cystic fibrosis (CF), the most common life-threatening inherited disorder among White individuals. Current CFTR correctors and potentiators, such as elexacaftor-tezacaftor-ivacaftor (ETI), only partially restore the function of the most prevalent mutant, F508del-CFTR, resulting in residual disease in people with CF. Here, we demonstrate that a mimetic peptide targeting the A-kinase–anchoring protein (AKAP) function of PI3Kγ (PI3Kγ MP), and driving localized cAMP elevation, enhances F508del-CFTR membrane localization, maximizing ETI efficacy in restoring chloride secretion. Mechanistically, PI3Kγ MP activates an AKAP-Lbc–anchored pool of PKD1, a known regulator of membrane trafficking. Consistently, PKD1 inhibition prevents PI3Kγ MP from enhancing the membrane expression of ETI-corrected F508del-CFTR. Overall, our findings reveal a regulatory pathway controlling CFTR membrane abundance via the AKAP function of PI3Kγ, which can be targeted to overcome the limitations of current CFTR modulator therapies.

Authors

Alessandra Murabito, Marco Mergiotti, Valeria Capurro, Alessia Loffreda, Mingchuan Li, Paola Peretto, Kai Ren, Andrea Raimondi, Carlo Tacchetti, Dario Diviani, Nicoletta Pedemonte, Emilio Hirsch, Alessandra Ghigo

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Figure 4

PI3Kγ MP activates AKAP-Lbc–anchored PKD1.

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PI3Kγ MP activates AKAP-Lbc–anchored PKD1. (A and B) Representative West...
(A and B) Representative Western blot (A) and relative quantification (B) of phospho-PKD1 (pPKD1 S744-748) in HEK293T cells overexpressing PKD1 and treated with CP or PI3Kγ MP (25 μM). Poly-L-arginine (5 μM, 10 minutes) served as positive control. n = 3. (C) Proposed mechanism of PKD1 activation: PI3Kγ MP elevates cAMP levels, leading to PKA activation, phosphorylation of AKAP-Lbc, and full activation and release of PKD1 from signalosome. (D and E) Representative Western blot (D) and relative quantification (E) of pPKD1 S744-748 in HEK293T cells overexpressing FLAG-PKD1 and treated with PI3Kγ MP (25 μM, 30 minutes) ± PKC inhibitor GO6983 (0.5 μM, 30 min preincubation). n = 4. (F and G) Representative Western blot (F) and relative quantification (G) of phosphorylated AKAP-Lbc in HEK293T cells overexpressing GFP-AKAP-Lbc and treated with vehicle, PI3Kγ MP (25 μM, 30 minutes), or Fsk (1.5 μM, 10 minutes; positive control). AKAP-Lbc was immunoprecipitated and IP pellets probed with PKA substrate antibody. TL, total lysate. n = 3. (H and I) Representative Western blot (H) and relative quantification (I) of PKD1 bound to AKAP-Lbc in HEK293T cells overexpressing GFP-AKAP-Lbc and FLAG-PKD1, treated with vehicle, PI3Kγ MP, or forskolin as in F. n = 3. (J and K) Representative Western blot (J) and relative quantification (K) of pPKD1 S744-748 in HEK293T cells overexpressing FLAG-PKD1 alone or with AKAP-Lbc PH domain, treated with vehicle or PI3Kγ MP (25 μM, 30 minutes). n = 4. In B, ##P < 0.01 for PI3Kγ MP at t = 30 versus t = 0 minutes; *P < 0.05 PI3Kγ MP versus CP at t = 30 minutes by 2-tailed Student’s t test. In G and I, *P < 0.05 PI3Kγ MP versus vehicle by Student’s t test. In E and K, *P < 0.05, **P < 0.01 by 1-way ANOVA with Dunnett’s multiple-comparison test. Data shown as mean ± SEM; n = independent experiments; data points = independent biological replicates.