Selective Regulation of Hepatic Protein Delivery: Mechanisms and Binding Partners of MAL2
Most classes of newly synthesized hepatic apical proteins take an indirect pathway to the cell surface. They are delivered from the TGN to the basolateral domain, selectively internalized then transcytosed to the apical surface. MAL2 has been implicated in regulating at least two steps in this pathway. MAL2 was first identified as a hepatic transcytotic regulator that mediates delivery from basolateral endosomes to the sub-apical compartment (SAC). However, overexpression of polymeric immunoglobulin A-receptor (pIgA-R) in polarized, hepatic WIF-B cells led to the dramatic redistribution of MAL2 into the Golgi and all the transcytotic intermediates occupied by the receptor. Although overexpressed hemagglutinin and dipeptidylpeptidase IV (DPPIV) distributed to the same compartments, MAL2 distributions did not change indicating the effect is selective. We found that DPPIV distributions were independent of MAL2, but surface delivery of pIgA-R was dependent on MAL2 expression. Thus, in addition to its role in transcytosis, MAL2 also regulates pIgA-R delivery from the Golgi to the basolateral membrane. Our studies have also shown that MAL2 and pIgA-R (but not DPP IV) selectively coimmunoprecipitate, but that their interactions are likely weak, transient, or indirect, suggesting other proteins are required to direct pIgA-R along its cellular itinerary. Because vesicle formation and delivery are driven by complex machineries, we predicted that MAL2 exists in large multi-protein complexes. To identify MAL2 interactors, we performed a split-ubiquitin yeast two hybrid screen using human MAL2 as bait. From a human liver cDNA library, serine-threonine kinase 16 (STK16) was identified. This lipid-anchored kinase is enriched in liver and shown to regulate mammary gland development implicating it as a likely candidate for regulating polarized protein trafficking. While overexpression of STK16 did not disrupt normal trafficking processes, we found that expression of the mutant, kinase-dead form of STK16 (KD-STK16) rerouted the secretory proteins albumin and haptoglobin from the secretory pathway to the degradative lysosomal pathway. Knockdown of MAL2 caused the same defect, implicating the interaction of MAL2 and STK16 as regulators of the secretory pathway. Based on our previous research with MAL2, it is suggested that MAL2 is an essential component of multiple trafficking pathways in epithelial cells whose activity must be tightly controlled to ensure proper polarity maintenance and growth.
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