Major Cdk5-dependent phosphorylation sites of amphiphysin 1 are implicated in the regulation of the membrane binding and endocytosis.
Amphiphysin 1 (amph 1) is an endocytic protein enriched in the nerve terminals that functions in the clathrin-mediated endocytosis. It acts as membrane curvature sensor, a linker of clathrin coat proteins, and an enhancer of dynamin Guanosine Triphosphatase (GTPase) activity. Amph 1 undergoes phosphorylation by cyclin-dependent kinase 5 (Cdk5), at five phosphorylation sites, serine 262, 272, 276, 285, and threonine 310, as determined by mass spectrometry (MS). We show here that Cdk5-dependent phosphorylation of amph 1 is enhanced in the presence of lipid membranes. Analysis by tandem liquid chromatograph MS revealed that the phosphorylation occurs at two phosphorylation sites. The phosphorylation was markedly decreased by mutation either Ser276 or Ser285 of amph 1 to alanine (S276A and S285A). Furthermore, mutation of both sites (S276, 285A) completely eliminated the phosphorylation. Functional studies indicated that binding of amph 1 to lipid membrane was attenuated by Cdk5-dependent phosphorylation of wild type amph 1, but not of the S276, 285A form. Interestingly, endocytosis was increased in rat pheochromocytoma cells expressing amph 1 S276, 285A in comparison with wild type. These results suggest that Ser276 and Ser285 are regulatory Cdk5 phosphorylation sites of amph 1 in the lipid-bound state. Phosphorylation at these sites alters binding of amph 1 to lipid membranes, and may be an important regulatory aspect in the regulation of synaptic vesicle endocytosis.
Liang
Shuang
S
Departments of NeurosciencePhysiology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, JapanLaboratory of Seeds Finding Technology, Eisai Co., Ltd., Ibaraki, Japan.
Wei
Fan-Yan
FY
Departments of NeurosciencePhysiology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, JapanLaboratory of Seeds Finding Technology, Eisai Co., Ltd., Ibaraki, Japan.
Wu
Yu-Mei
YM
Departments of NeurosciencePhysiology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, JapanLaboratory of Seeds Finding Technology, Eisai Co., Ltd., Ibaraki, Japan.
Tanabe
Kenji
K
Departments of NeurosciencePhysiology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, JapanLaboratory of Seeds Finding Technology, Eisai Co., Ltd., Ibaraki, Japan.
Abe
Tadashi
T
Departments of NeurosciencePhysiology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, JapanLaboratory of Seeds Finding Technology, Eisai Co., Ltd., Ibaraki, Japan.
Oda
Yoshiya
Y
Departments of NeurosciencePhysiology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, JapanLaboratory of Seeds Finding Technology, Eisai Co., Ltd., Ibaraki, Japan.
Yoshida
Yumi
Y
Departments of NeurosciencePhysiology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, JapanLaboratory of Seeds Finding Technology, Eisai Co., Ltd., Ibaraki, Japan.
Yamada
Hiroshi
H
Departments of NeurosciencePhysiology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, JapanLaboratory of Seeds Finding Technology, Eisai Co., Ltd., Ibaraki, Japan.
Matsui
Hideki
H
Departments of NeurosciencePhysiology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, JapanLaboratory of Seeds Finding Technology, Eisai Co., Ltd., Ibaraki, Japan.
Tomizawa
Kazuhito
K
Departments of NeurosciencePhysiology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, JapanLaboratory of Seeds Finding Technology, Eisai Co., Ltd., Ibaraki, Japan.
Takei
Kohji
K
Departments of NeurosciencePhysiology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, JapanLaboratory of Seeds Finding Technology, Eisai Co., Ltd., Ibaraki, Japan.
D000409:Alanine; D000818:Animals; D051360:Cyclin-Dependent Kinase 5 / Q000494:pharmacology* / Q000502:physiology; D004347:Drug Interactions; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D008055:Lipids; D008081:Liposomes / Q000494:pharmacology; D013058:Mass Spectrometry; D008855:Microscopy, Electron, Scanning / Q000379:methods; D016296:Mutagenesis / Q000502:physiology; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism* / Q000493:pharmacokinetics; D016716:PC12 Cells; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011687:Purines / Q000494:pharmacology; D051381:Rats; D000077546:Roscovitine; D012694:Serine / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D013912:Threonine / Q000378:metabolism; D014162:Transfection
D008055:Lipids; D008081:Liposomes; D009419:Nerve Tissue Proteins; D047428:Protein Kinase Inhibitors; D011687:Purines; D000077546:Roscovitine; C075637:amphiphysin; D013912:Threonine; D012694:Serine; D051360:Cyclin-Dependent Kinase 5; D000409:Alanine