A Serine/Threonine Kinase 16-Based Phospho-Proteomics Screen Identifies WD Repeat Protein-1 As A Regulator Of Constitutive Secretion

Scientific Reports
Alfonso López-CoralP L Tuma

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...Continue Reading

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Citations

Oct 3, 2019·International Journal of Molecular Sciences·Junjun WangXin Zhang
Apr 13, 2019·International Journal of Molecular Sciences·Junjun WangXin Zhang

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Methods Mentioned

BETA
electrophoresis
transfection
two hybrid
GTPases
protein assay
Fluorescence

Software Mentioned

ImageJ
ProteinLynx Global Server ( PLGS
Progenesis Same Spots
Progenesis PG240
Mascot
Metamorph Imaging
Adobe Photoshop

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