Endocytosis of somatodendritic NCKX2 is regulated by Src family kinase-dependent tyrosine phosphorylation

Abstract

We have previously reported that the surface expression of K+-dependent Na+/Ca2+ exchanger 2 (NCKX2) in the somatodendritic compartment is kept low by constitutive endocytosis, which results in the polarization of surface NCKX2 to the axon. Clathrin-mediated endocytosis is initiated by interaction of the μ subunit of adaptor protein complex 2 (AP-2) with the canonical tyrosine motif (YxxΦ) of a target molecule. We examined whether endocytosis of NCKX2 involves two putative tyrosine motifs (365YGKL and 371YDTM) in the cytoplasmic loop of NCKX2. Coimmunoprecipitation assay revealed that the 365YGKL motif is essential for the interaction with the μ subunit of AP-2 (AP2M1). Consistently, either overexpression of NCKX2-Y365A mutant or knockdown of AP2M1 in cultured hippocampal neurons significantly reduced the internalization of NCKX2 from the somatodendritic surface and thus abolished the axonal polarization of surface NCKX2. Next, we tested whether the interaction between the tyrosine motif and AP2M1 is regulated by phosphorylation of the 365th tyrosine residue (Tyr-365). Tyrosine phosphorylation of heterologously expressed NCKX2-WT, but not NCKX2-Y365A, was increased by carbachol (CCh) in PC-12 cells. The effect of CCh was inhibited by PP2, a Src family kinase (SFK) inhibitor. Moreover, PP2 facilitated the endocytosis of NCKX2 in both the somatodendritic and axonal compartments, suggesting that tyrosine phosphorylation of NCKX2 by SFK negatively regulates its endocytosis. Supporting this idea, activation of SFK enhanced the NCKX activity in the proximal dendrites of dentate granule cells (GCs). These results suggest that endocytosis of somatodendritic NCKX2 is regulated by SFK-dependent phosphorylation of Tyr-365.

Inhibition of non‐receptor tyrosine kinase Src induces phosphoserine 256‐independent aquaporin‐2 membrane accumulation

“…When Src activity is blocked, AQP2 accumulates at the membrane, implying that Src plays a role in maintaining the balance of AQP2 constitutive recycling so that membrane accumulation does not normally occur under baseline conditions. It is perhaps not surprising that Src should play a role in AQP2 constitutive recycling, as Src has been previously implicated in the constitutive internalization of G-protein coupled receptors or channels (Chao et al 2005;Lee et al 2013). In our study, we were not able to measure a significant change in actin depolymerization (data not shown), which is believed to be an important step in AQP2 membrane accumulation (Noda et al 2008;Yui et al 2012), and the mechanism by which Src inhibition increases exocytosis remains elusive.…”

“…; Lee et al . ). In our study, we were not able to measure a significant change in actin depolymerization (data not shown), which is believed to be an important step in AQP2 membrane accumulation (Noda et al .…”

Multiple Calcium Export Exchangers and Pumps Are a Prominent Feature of Enamel Organ Cells

“…NCKX1 is expressed primarily in retinal rod photoreceptors and platelets (Schnetkamp, 2004 ; Lytton, 2007 ). NCKX2 is expressed in cone photoreceptors and is involved in mouse motor learning and memory (Schnetkamp, 2004 ; Lee et al, 2009 , 2013 ), and NCKX3 is expressed in the brain and the kidneys (Schnetkamp, 2004 ; Lee et al, 2009 ) though it is expressed in the kidneys at higher levels in female mice than in male mice (Lee et al, 2009 ). NCKX3 is also highly expressed in the human endometrium during the menstrual cycle, where its expression is partially regulated by the steroid hormone 17β-estradiol (Yang et al, 2011 ).…”

Bioinformatics-based study reveals that AP2M1 is regulated by the circRNA-miRNA-mRNA interaction network and affects Alzheimer’s disease

“…On my STRING website, we can observe that MAPT, GSK3B, and subunits of the AP-2 complex, including AP2M1, are closely related (Supplementary Material 3). Among them, AP2M1 and MAPT are co-expressed and some known experimentally determined interactions (Lee et al, 2013;Freeze et al, 2018). And they are mainly enriched in the GO terms, including Cell-cell signaling, Cellular protein metabolic process, Transport, Cellular component organization, and Wnt signaling pathway (Supplementary Material 4).…”