Interaction of furin in immature secretory granules from neuroendocrine cells with the AP-1 adaptor complex is modulated by casein kinase II phosphorylation.
The composition of secretory granules in neuroendocrine and endocrine cells is determined by two sorting events; the first in the trans-Golgi complex (TGN), the second in the immature secretory granule (ISG). Sorting from the ISG, which may be mediated by the AP-1 type adaptor complex and clathrin-coated vesicles, occurs during ISG maturation. Here we show that furin, a ubiquitously expressed, TGN/endosomal membrane endoprotease, is present in the regulated pathway of neuroendocrine cells where it is found in ISGs. By contrast, TGN38, a membrane protein that is also routed through the TGN/endosomal system does not enter ISGs. Furin, however, is excluded from mature secretory granules, suggesting that the endoprotease is retrieved from the clathrin-coated ISGs. Consistent with this, we show that the furin cytoplasmic domain interacts with AP-1, a component of the TGN/ISG-localized clathrin sorting machinery. Interaction between AP-1 and furin is dependent on phosphorylation of the enzyme's cytoplasmic domain by casein kinase II. Finally, in support of a requirement for the phosphorylation-dependent association of furin with AP-1, expression of furin mutants that mimic either the phosphorylated or unphosphorylated forms of the endoprotease in AtT-20 cells demonstrates that the integrity of the CKII sites is necessary for removal of furin from the regulated pathway.
Dittié
A S
AS
Secretory Pathways Laboratory, Imperial Cancer Research Fund, London, UK.
Thomas
L
L
Thomas
G
G
Tooze
S A
SA
D033965:Adaptor Protein Complex alpha Subunits; D033942:Adaptor Proteins, Vesicular Transport; D000469:Alkaline Phosphatase / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D001667:Binding, Competitive; D047390:Casein Kinase II; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D002966:Clathrin / Q000378:metabolism; D003001:Cloning, Molecular; D003594:Cytoplasmic Granules / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D045683:Furin; D006023:Glycoproteins; D006056:Golgi Apparatus / Q000378:metabolism; D015151:Immunoblotting; D008562:Membrane Glycoproteins / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D016716:PC12 Cells; D010766:Phosphorylation; D011233:Precipitin Tests; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins; D013381:Subtilisins / Q000378:metabolism*
D033965:Adaptor Protein Complex alpha Subunits; D033942:Adaptor Proteins, Vesicular Transport; D002966:Clathrin; D006023:Glycoproteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; C513045:Tgoln2 protein, rat; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D000469:Alkaline Phosphatase; D013381:Subtilisins; D045683:Furin