Phosphatidic acid‐PKA signaling regulates p38 and ERK1/2 functions in ligand‐independent EGFR endocytosis

Abstract

Ligand-independent epidermal growth factor receptor (EGFR) endocytosis is inducible by a variety of stress conditions converging upon p38 kinase. A less known pathway involves phosphatidic acid (PA) signaling toward the activation of type 4 phosphodiesterases (PDE4) that decrease cAMP levels and protein kinase A (PKA) activity. This PA/PDE4/PKA pathway is triggered with propranolol used to inhibit PA hydrolysis and induces clathrin-dependent and clathrin-independent endocytosis, followed by reversible accumulation of EGFR in recycling endosomes. Here we give further evidence of this signaling pathway using biosensors of PA, cAMP, and PKA in live cells and then show that it activates p38 and ERK1/2 downstream the PKA inhibition. Clathrin-silencing and IN/SUR experiments involved the activity of p38 in the clathrin-dependent route, while ERK1/2 mediates clathrin-independent EGFR endocytosis. The PA/PDE4/PKA pathway selectively increases the EGFR endocytic rate without affecting LDLR and TfR constitute endocytosis. This selectiveness is probably because of EGFR phosphorylation, as detected in Th1046/1047 and Ser669 residues. The EGFR accumulates at perinuclear recycling endosomes colocalizing with TfR, fluorescent transferrin, and Rab11, while a small proportion distributes to Alixendosomes. A non-selective recycling arrest includes LDLR and TfR in a reversible manner. The PA/PDE4/PKA pathway involving both p38 and ERK1/2 expands the possibilities of EGFR transmodulation and interference in cancer.

Phosphorylation and ubiquitination independent endocytosis of BRI1

“…Similar observations were reported for the borate exporter BOR1, in which AP-2-dependent and AP-2-independent endocytic routes had been activated by low and high borate concentration, respectively (Yoshinari et al, 2019). In mammals, the well-studied epidermal growth factor receptor (EGFR) is also internalized via multiple endocytic routes, including canonical ligand-dependent clathrin-mediated endocytosis and clathrin-independent endocytosis route, which both depend on the ligand concentration (Zhou and Sakurai, 2022), and a ligand-independent route where EGFR endocytosis is induced by stress conditions and does not require kinase activity or ubiquitination (Metz et al, 2021). Taken together, our results show that the BRI1 internalization is not abolished in the BR binding-deficient mutant, similarly to BRI1 YXXΦ endocytic motif mutants (Liu et al, 2020), the BRI1 ubiquitination-deficient mutants (Martins et al, 2015; Zhou et al, 2018) and the BRI1 endocytosis in AP-2 mutants(Di Rubbo et al, 2013; Gadeyne et al, 2014).…”

Phosphatidic acid: from biophysical properties to diverse functions

“…It was reported that the internalization of inactive EGFR is through a PA effector‐rolipram‐sensitive type 4 phosphodiesterase (PDE4) that mediated down‐regulation of protein kinase A (PKA) activity. The PA/PDE4/PKA pathway regulates p38 and ERK1/2 functions in ligand‐independent EGFR endocytosis [28]. PLD2 activity has been shown to be important for internalization of micro‐opioid receptors and metabotropic glutamate receptors [3].…”

BRASSINOSTEROID INSENSITIVE1 internalization can occur independent of ligand binding

“…Similar observations were reported for the borate exporter BOR1, in which AP-2-dependent and AP-2independent internalization had been activated by low and high borate concentration, respectively (Yoshinari et al, 2019). Equally in mammals, the well-studied epidermal growth factor receptor (EGFR) is also internalized via different endocytic mechanisms, including canonical liganddependent clathrinmediated endocytosis and clathrin-independent endocytosis, which both depend on the ligand concentration (Zhou and Sakurai, 2022), and a ligand-independent internalization where EGFR endocytosis is induced by stress conditions and does not require kinase activity or ubiquitination (Metz et al, 2021). However, after internalization, BRI1 and BRI1 Q pursued the same trafficking routes.…”