Alpha-kinase 1, a new component in apical protein transport

J Biol Chem. 2005 Jul 8;280(27):25637-43. doi: 10.1074/jbc.M502265200. Epub 2005 May 9.

Abstract

A key aspect in the structure of epithelial cells is the maintenance of a polarized organization based on a highly specific sorting machinery for cargo destined for the apical or the basolateral membrane domain at the exit site of the trans-Golgi network. We could recently identify two distinct post-trans-Golgi network vesicle populations that travel along separate routes to the plasma membrane, a lipid raft-dependent and a lipid raft-independent pathway. A new component of raft-carrying apical vesicles is alpha-kinase 1 (ALPK1), which was identified in immunoisolated vesicles carrying raft-associated sucrase-isomaltase (SI). This kinase was absent from vesicles carrying raft-non-associated lactase-phlorizin hydrolase. The expression of ALPK1 increases by the time of epithelial cell differentiation, whereas the intracellular localization of ALPK1 on apical transport vesicles was confirmed by confocal analysis. A phosphorylation assay on isolated SI-carrying vesicles revealed the phosphorylation of a protein band of about 105 kDa, which could be identified as the motor protein myosin I. Finally, a specific reduction of ALPK1-expression by RNA interference results in a significant decrease in the apical delivery of SI. Taken together, our data suggest that the phosphorylation of myosin I by ALPK1 is an essential process in the apical trafficking of raft-associated SI.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • COS Cells
  • Caco-2 Cells
  • Cell Membrane / enzymology
  • Cell Polarity / physiology*
  • Chlorocebus aethiops
  • Dogs
  • Epithelial Cells / enzymology*
  • Exocytosis / physiology
  • Humans
  • Isomaltose / metabolism
  • Kidney / cytology
  • Membrane Microdomains / enzymology*
  • Myosin Type I / metabolism
  • Protein Kinase Inhibitors / pharmacology
  • Protein Kinases / genetics
  • Protein Kinases / metabolism*
  • Protein Transport / physiology
  • Staurosporine / pharmacology
  • trans-Golgi Network / enzymology

Substances

  • Protein Kinase Inhibitors
  • Isomaltose
  • Protein Kinases
  • Myosin Type I
  • Staurosporine