AKAP350 Is involved in the development of apical “Canalicular” structures in hepatic cells HepG2

Abstract

Hepatocytes are epithelial cells whose apical poles constitute the bile canaliculi. The establishment and maintenance of canalicular poles is a finely regulated process that dictates the efficiency of primary bile secretion. Protein kinase A (PKA) modulates this process at different levels. AKAP350 is an A-kinase anchoring protein that scaffolds protein complexes involved in modulating the dynamic structures of the Golgi apparatus and microtubule cytoskeleton, facilitating microtubule nucleation at this organelle. In this study, we evaluated whether AKAP350 is involved in the development of bile canaliculi-like structures in hepatocyte derived HepG2 cells. We found that AKAP350 recruits PKA to the centrosomes and Golgi apparatus in HepG2 cells. De-localization of AKAP350 from these organelles led to reduced apical cell polarization. A decrease in AKAP350 expression inhibited the formation of canalicular structures and impaired F-actin organization at canalicular poles. Furthermore, loss of AKAP350 expression led to diminished polarized expression of the p-glycoprotein (MDR1/ABCB1) at the apical “canalicular” membrane. AKAP350 knock down effects on canalicular structures formation and actin organization could be mimicked by inhibition of Golgi microtubule nucleation by depletion of CLIP associated proteins (CLASPs). Our data reveal that AKAP350 participates in mechanisms which determine the development of canalicular structures as well as accurate canalicular expression of distinct proteins and actin organization, and provide evidence on the involvement of Golgi microtubule nucleation in hepatocyte apical polarization.

RSK1 protects P-glycoprotein/ABCB1 against ubiquitin–proteasomal degradation by downregulating the ubiquitin-conjugating enzyme E2 R1

“…PKA isoform, type II (PKA-RIIα) and A-kinase anchoring protein (AKAP350) modulate P-gp trafficking to the apical canalicular membrane, and PKC regulates cell volume-activated chlorine channels through P-gp phosphorylation303132. These kinases phosphorylate a couple of serine residues in the intracellular linker region of P-gp, and the PP5-PPP2R3C complex negatively regulates P-gp expression by dephosphorylating these sites29.…”

Golgi as an MTOC: making microtubules for its own good

“…It is possible that the role of Golgi-derived MTs in cell polarity is abundant in other cell types yet unexamined in this regard. For example, depletion of either AKAP or CLASPs in hepatocytes strongly diminishes structural and biochemical polarization of these cells during bile canaliculi morphogenesis (Mattaloni et al 2012). …”

Centrosomal AKAP350 and CIP4 act in concert to define centrosome/Golgi polarity in migratory cells

“…We prepared HepG2 AKAP350KD cells, as we have previously described (Mattaloni et al, 2012), and first evaluated the impact of the decrease in AKAP350 expression on HepG2 cell migration in wound healing assays. We then verified that, as previously described in melanoma cells, the cell migration efficiency of HepG2 AKAP350KD cells was impaired (Fig.…”

“…In order to reduce protein expression in HepG2 cells, specific 21-nucleotide double-chain RNA [small interfering (si)RNA] and a scrambled control were designed, as we have previously described (Mattaloni et al, 2012), and synthesized using an Ambion commercial kit 'SilencerTMsiRNA'. The following sequences targeting AKAP350 or CIP4 mRNA were used -AKAP350 siRNA1 5′-AAATCCCTTGCCAGCACATGA-3′, AKAP350 siRNA2 5′-AAGCAAGAACTAGAACGAGAA-3′ and CIP4 siRNA 5′-GACCTCAGTCTTATGGAAGAA-3′.…”

“…Populations of cells that stably expressed AKAP350CTD-GFP or GFP (control) were generated. HepG2 cells were transfected by using electroporation, as previously described (Mattaloni et al, 2012). Sk-Hep1 and MDCK cells were transfected using Amaxa Nucleofector™ 2b with program X-001.…”