Intracellular trafficking of furin is modulated by the phosphorylation state of a casein kinase II site in its cytoplasmic tail.
Human furin catalyzes the proteolytic maturation of many proproteins in the exocytic and endocytic secretory pathways by cleavage at the C-terminal side of the consensus sequence-ArgXaaLys/ArgArg decreases -. Both the trans-Golgi network (TGN) concentration and intracellular routing of furin require sequences in its 56 amino acid cytoplasmic tail. Here, we show that the furin cytoplasmic tail contains multiple trafficking signals. Localization to the TGN requires a cluster of acidic amino acids that, together with a pair of serine residues, forms a casein kinase II (CK II) phosphorylation site. We show that CK II efficiently phosphorylates these serines in vitro, and using a permeabilized cell system we provide evidence that CK II is the in vivo furin kinase. Analysis by mass spectrometry shows that, in vivo, furin exists as di-, mono- and non-phosphorylated forms. Finally, employing (i) furin constructs that mimic either non-phosphorylated or phosphorylated furin and (ii) the phosphatase inhibitor tautomycin, we show that the phosphorylation state of the furin cytoplasmic tail modulates retrieval of the endoprotease to the TGN. Thus, routing of furin is a two-tiered process combining a set of trafficking signals comprised of the primary amino acid sequence of the tail with its phosphorylation state.
Jones
B G
BG
Vollum Institute, Oregon Health Sciences University, Portland 97201-3098, USA.
Thomas
L
L
Molloy
S S
SS
Thulin
C D
CD
Fry
M D
MD
Walsh
K A
KA
Thomas
G
G
D000595:Amino Acid Sequence; D047390:Casein Kinase II; D002460:Cell Line; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism; D011992:Endosomes / Q000201:enzymology / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D045683:Furin; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational / Q000235:genetics; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012694:Serine / Q000378:metabolism; D013381:Subtilisins / Q000737:chemistry / Q000378:metabolism*; D014168:Transferrin / Q000378:metabolism
D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D014168:Transferrin; D012694:Serine; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D010744:Phosphoric Monoester Hydrolases; D013381:Subtilisins; D045683:Furin