Anchoring of Protein Kinase A-Regulatory Subunit IIα to Subapically Positioned Centrosomes Mediates Apical Bile Canalicular Lumen Development in Response to Oncostatin M but Not cAMP

Abstract

Oncostatin M and cAMP signaling stimulate apical surface-directed membrane trafficking and apical lumen development in hepatocytes, both in a protein kinase A (PKA)-dependent manner. Here, we show that oncostatin M, but not cAMP, promotes the A-kinase anchoring protein (AKAP)-dependent anchoring of the PKA regulatory subunit (R)II␣ to subapical centrosomes and that this requires extracellular signal-regulated kinase 2 activation. Stable expression of the RIIdisplacing peptide AKAP-IS, but not a scrambled peptide, inhibits the association of RII␣ with centrosomal AKAPs and results in the repositioning of the centrosome from a subapical to a perinuclear location. Concomitantly, common endosomes, but not apical recycling endosomes, are repositioned from a subapical to a perinuclear location, without significant effects on constitutive or oncostatin M-stimulated basolateral-to-apical transcytosis. Importantly, however, the expression of the AKAP-IS peptide completely blocks oncostatin M-, but not cAMP-stimulated apical lumen development. Together, the data suggest that centrosomal anchoring of RII␣ and the interrelated subapical positioning of these centrosomes is required for oncostatin M-, but not cAMP-mediated, bile canalicular lumen development in a manner that is uncoupled from oncostatin M-stimulated apical lumen-directed membrane trafficking. The results also imply that multiple PKA-mediated signaling pathways control apical lumen development and that subapical centrosome positioning is important in some of these pathways. INTRODUCTIONPolarized hepatocytes, like all epithelial cells, display distinct plasma membrane domains, an apical plasma membrane domain facing the bile canalicular lumen, and a basolateral plasma membrane domain facing the space of Disse. Concomitant with cell surface polarity, also the cell interior displays a polarized organization. A dense cortical actin network is assembled beneath the apical surface and actin filaments extend into apical microvilli with their barbed ends facing the microvilli tips. In addition, part of the microtubule cytoskeleton is oriented parallel to the apicalbasolateral axis with their minus and plus ends facing the apical and basolateral surface, respectively. The cytoskeleton organization influences the position of the centrosome (Burakov et al., 2003), which in various epithelial cells including intestinal Caco-2, kidney Madin-Darby canine kidney (MDCK), and hepatic WIF-B cells, is typically oriented toward the apical surface (Buendia et al., 1990;Meads and Schroer, 1995;Salas, 1999). Polarized positioning of the centrosome is thought to play a role in the establishment of epithelial cell polarity (Zeligs, 1979;Dylewski and Keenan, 1984;Houliston et al., 1987;Rizzolo and Joshi, 1993) and neuronal polarity (de Anda et al., 2005;Higginbotham et al., 2006). A subapical position of the centrosome may be important for the polarized orientation of microtubule-associated organelles such as the Golgi apparatus and recycling endosomes, and, in this way, facilitate the...

cAMP‐dependent protein kinase A and the dynamics of epithelial cell surface domains: Moving membranes to keep in shape

“…Stimulation of hepatocytes with OSM does not elevate cAMP levels or stimulate overall PKA activity but enhances the association of PKA-RIIα with centrosomes in an ERK1-dependent manner (80,103) . 18 Displacement of PKA-RIIα from the centrosome, by means of a small interfering peptide (80) or by forcing synchronised hepatocytes into the S-phase of the cell cycle (104) , prevents the stimulatory effect of OSM on apical membrane biogenesis.…”

“…repositioning of the centrosome and surrounding recycling endosomes (80) . In the latter study, surprisingly, inhibition of catalytic PKA activity did not alter the position of the centrosome and recycling endosomes.…”

Apical trafficking in epithelial cells: signals, clusters and motors

“…Interestingly, a different kinesin is required for p75 transport to the PM in non-polarized MDCK cells (Geri Kreitzer, personal communication). The diversity of MT motors that participate in apical trafficking might explain puzzling observations in MDCK cell variants and hepatocytes that fail to reorganize their MTs during polarization but nonetheless can polarize normally under such conditions (Grindstaff et al, 1998;Wojtal et al, 2007) Cooperation of MT motors and the actin cytoskeleton in apical trafficking…”

Formation of E-Cadherin/β-Catenin–based Adherens Junctions in Hepatocytes Requires Serine-10 in p27(Kip1)

“…Pretreatment of the cells with PD98059 (50 M), which effectively inhibits the activation of p42/44 MAPK by MAPK kinase in response to oncostatin M (Wojtal et al, 2007), prevents the oncostatin M-stimulated increase in p27-Ser10 phosphorylation ( Figure 1A). In addition, PD98059 prevents the stimulatory effect of oncostatin M on cell-cell adhesion ( Figure 1C).…”

“…This effect of OSM on adherens junction formation is believed to be part of the complex mechanism by which OSM signaling controls liver development (Kamiya et al, 1999;Kinoshita and Miyajima, 2002), which also includes the establishment of apical-basolateral polarity (van der Wouden et al, 2002;Wojtal et al, 2007;van IJzendoorn et al, 2004). OSM, via the signal transducer protein gp130, typically stimulates two major signaling pathways that involve Ras/mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription (STAT; Heinrich et al, 1998).…”