Abstract
The plasma membrane of polarized hepatocytes is functionally divided into two domains: the apical and basolateral. Our focus is to define the molecular basis of polarized protein sorting of newly-synthesized membrane and secretory proteins in WIF-B cells, an excellent model system for polarized hepatocytes. We determined that MAL2 (myelin and lymphocyte protein 2) and its binding partner, serine/threonine kinase 16 (STK16) regulate basolateral constitutive secretion. Because STK16 is a constitutively active kinase, we reasoned that constitutively phosphorylated substrates must participate in constitutive secretion. To identify either STK16 substrates or other proteins that regulate constitutive secretion, we took a proteomics approach. Post-nuclear supernatants from cells expressing wild type or a kinase-dead (E202A) STK16 were separated on 2D gels and immunoblotted with antibodies against phospho-serine/threonine residues. Sixteen spots were identified from E202A-expressing cells that reproducibly displayed decreased immunoreactivity. From these spots, 28 proteins were identified as possible STK16 substrates. Out of these 28 possible substrates, 25% of them encode predicted STK16 phosphorylation consensus sites, with WD repeat containing protein-1 (WDR1) encoding two such sites. Based on this finding and on the finding that actin remodeling is required for hepatic secretion, we further confirmed that WDR1 is a phosphoprotein that regulates secretion.
Highlights
IntroductionWe initiated studies to examine the role of MAL2 (myelin and lymphocyte protein 2) in regulating hepatic polarized protein sorting
Over a decade ago, we initiated studies to examine the role of MAL2 in regulating hepatic polarized protein sorting
Based on the finding that actin remodeling is required for hepatic secretion[35], we further examined WD repeat-containing protein 1(WDR1) 1 as a potential component of the molecular machinery that drives constitutive secretion or as a part of a serine/threonine kinase 16 (STK16)-mediated phosphoprotein network that regulates secretion
Summary
We initiated studies to examine the role of MAL2 (myelin and lymphocyte protein 2) in regulating hepatic polarized protein sorting. Based on our previous results of the overexpression of a dominant negative, kinase-dead version of the enzyme, we determined that kinase activity is required for STK16 function in secretion[33]. This further implies that constitutively phosphorylated substrates must participate in constitutive secretion. Based on the finding that actin remodeling is required for hepatic secretion[35], we further examined WD repeat-containing protein 1(WDR1) 1 as a potential component of the molecular machinery that drives constitutive secretion or as a part of a STK16-mediated phosphoprotein network that regulates secretion
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