Essential role of flotillin-1 palmitoylation in the intracellular localization and signaling function of IGF-1 receptor

Abstract

Here, we explored flotillin-1-mediated regulation of insulin-like growth factor-1 (IGF-1) signaling. Flotillin-1-deficient cells exhibited a reduction in the activation of IGF-1 receptor (IGF-1R), ERK1/2 and Akt pathways, and the transcriptional activation of Elk-1 and the proliferation in response to IGF-1 were reduced in these cells. We found that IGF-1-independent flotillin-1 palmitoylation at Cys34 in the endoplasmic reticulum (ER) was required for the ER exit and the plasma membrane localization of flotillin-1 and IGF-1R. IGF-1-dependent depalmitoylation and repalmitoylation of flotillin-1 sustained tyrosine kinase activation of the plasma-membranetargeted IGF-1R. Dysfunction and blocking the turnover of flotillin-1 palmitoylation abrogated cancer cell proliferation after IGF-1R signaling activation. Our data show that flotillin-1 palmitoylation is a new mechanism by which the intracellular localization and activation of IGF-1R are controlled.

Flotillins: At the Intersection of Protein S-Palmitoylation and Lipid-Mediated Signaling

“…Until now, only an S-palmitoylation-dependent plasma membrane localization of flotillin-1 has been confirmed. In several cell lines, overexpressed wild-type flotillin-1 localized to the plasma membrane while a non-palmitoylated Cys34Ala mutant displayed intracellular location [24,38,60], although contrary results have also been reported [69]. Interestingly, inhibition of protein S-palmitoylation induces a shift of endogenous flotillin-1 from the plasma membrane to intracellular vesicles [82].…”

“…EGF receptor signaling leading to cell adhesion [8] EGF receptor clustering and phosphorylation, ERK1/2 and Akt phosphorylation; flotillins as MAP kinase scaffolding proteins [9] EGF receptor expression in breast cancer cells [10] activation of H-Ras in breast cancer cells [11] EGF receptor sorting and lysosomal degradation [12] TLR3 ligand internalization [13] TCR raft association and recycling [14][15][16][17] PrP [Ca +2 ] increase, Fyn and ERK1/2 activation, N-cadherin trafficking [18,19] IgE receptor [Ca +2 ] increase, phosphorylation of IgE receptor γ chain and ERK1/2 [20] insulin receptor insulin-induced glucose uptake via Glut4 transporter [21] Gq protein-coupled receptors p38 phosphorylation [22] not determined signaling leading to axon regeneration: Rho GTPase activation, formation of N-WASP-Arp3/cortactin complexes, p38, ERK1/2 and FAK kinase phosphorylation [23] IGF-1 receptor* IGF-1 receptor transport from endoplasmic reticulum to plasma membrane [24] integrins ERK2 and FAK kinase phosphorylation [25] ACTIN CYTOSKELETON REMODELING Affected processes filopodia formation [26] cell spreading [8] proper localization of Vav during T cell spreading [14] F-actin binding [27] axon regeneration [23] uropod formation in neutrophils [28] uropod formation in T cells, activation of ezrin/radix/moesin, localization of PIP5KIγ to uropod [29][30][31] formation of lamellipodia at the growth cone of neurons [32] cell adhesion and migration via α-actinin binding [25] ENDOCYTOSIS Cargo fluid phase (magnetic dextran, AF488-dextran) [33,34] GPI-anchored proteins [18,33,[35]…”

“…Notably, the pattern of acylation is different for the two flotillins ( Figure 1). Flotillin-1 is S-palmitoylated on Cys34 located within the first hydrophobic stretch of the protein [24,59,60]. The potential palmitoylation of Cys5 and Cys17 predicted by relevant algorithms has not been confirmed experimentally (https://swisspalm.org).…”

“…Besides acylation, oligomerization is another factor affecting the membrane binding of flotillins. Flotillins exist as a mixture of monomers and homo-and/or hetero-tetramers, as indicated by chemical cross-linking and by sucrose density gradient fractionation of endogenous proteins [24,62]. Homooligomerization of flotillin-2 was also indicated by the yeast two-hybrid system [26].…”

Targeted enhancement of flotillin-dependent endocytosis augments cellular uptake and impact of cytotoxic drugs

“…Flotillins themselves are palmitoylated 15,54,55 , and DHHC palmitoylates flotillin-2 31 , suggesting that direct and specific palmitoylation of flotillins by DHHC5 could enhance fluid phase endocytosis upon USMB treatments. This hypothesis is difficult to test experimentally, as perturbation of flotillin palmitoylation by sitedirected mutagenesis of flotillins can result in broader alterations in membrane traffic of these proteins 56 . DHHC5 has several identified substrates and as such it is possible that USMB triggers the palmitoylation of many cell surface proteins, as was suggested to occur in MEND 26 .…”