Interaction with receptor for activated C-kinase 1 (RACK1) sensitizes the phosphodiesterase PDE4D5 towards hydrolysis of cAMP and activation by protein kinase C

We have previously identified the PKC (protein kinase C)-anchoring protein RACK1 (receptor for activated C-kinase 1), as a specific binding partner for the cAMP-specific phosphodiesterase PDE4D5, suggesting a potential site for cross-talk between the PKC and cAMP signalling pathways. In the present study we found that elevation of intracellular cAMP, with the β2-adrenoceptor agonist isoproterenol (isoprenaline), led to activation of PDE4 enzymes in the particulate and soluble fractions of HEK (human embryonic kidney)-293 cells. In contrast activation of PDE4D5, with isoproterenol and the PKC activator PMA, was restricted to the particulate fraction, where it interacts with RACK1; however, RACK1 is dispensable for anchoring PDE4D5 to the particulate fraction. Kinetic studies demonstrated that RACK1 alters the conformation of particulate-associated PDE4D5 so that it more readily interacts with its substrate cAMP and with rolipram, a PDE4 inhibitor that specifically targets the active site of the enzyme. Interaction with RACK1 was also essential for PKC-dependent and ERK (extracellular-signal-regulated kinase)-independent phosphorylation (on Ser126), and activation of PDE4D5 in response to PMA and isoproterenol, both of which trigger the recruitment of PKCα to RACK1. Together these results reveal novel signalling cross-talk, whereby RACK1 mediates PKC-dependent activation of PDE4D5 in the particulate fraction of HEK-293 cells in response to elevations in intracellular cAMP

Pacsin2 is required for endocytosis in the zebrafish pronephric tubule

Endocytosis mediates the cellular uptake of numerous molecules from the extracellular space and is a fundamentally important process. In the renal proximal tubule, the scavenger receptor megalin and its co-receptor cubilin mediate endocytosis of low molecular weight proteins from the renal filtrate. However, the extent to which megalin endocytosis relies on different components of the trafficking machinery remains relatively poorly defined in vivo. In this study, we identify a functional requirement for the F-BAR protein pacsin2 in endocytosis in the renal proximal tubule of zebrafish larvae. Pacsin2 is expressed throughout development and in all zebrafish tissues, similar to the mammalian orthologue. Within renal tubular epithelial cells, pacsin2 is enriched at the apical pole where it is localised to endocytic structures. Loss of pacsin2 results in reduced endocytosis within the proximal tubule, which is accompanied by a reduction in the abundance of megalin and endocytic organelles. Our results indicate that pacsin2 is required for efficient endocytosis in the proximal tubule, where it likely cooperates with other trafficking machinery to maintain endocytic uptake and recycling of megalin

Endosomal recycling tubule scission and integrin recycling involve the membrane curvature supporting protein LITAF

Recycling to the cell surface requires the scission of tubular membrane intermediates emanating from endosomes. Here, we identify the monotopic membrane protein LPS-induced TNF-activating factor (LITAF) and the related protein cell death involved p53 target 1 (CDIP1) as novel membrane curvature proteins that contribute to recycling tubule scission. Recombinant LITAF supports high membrane curvature, shown by its ability to reduce proteoliposome size. The membrane domains of LITAF and CDIP1 partition strongly into ∼50 nm diameter tubules labelled with the recycling markers Pacsin2, ARF6 and SNX1, and the recycling cargoes MHC class I and CD59. Partitioning of LITAF into tubules is impaired by mutations linked to Charcot Marie Tooth disease type 1C. Meanwhile, co-depletion of LITAF and CDIP1 results in the expansion of tubular recycling compartments and stabilised Rab11 tubules, pointing to a function for LITAF and CDIP1 in membrane scission. Consistent with this, co-depletion of LITAF and CDIP1 impairs integrin recycling and cell migration