Major Cdk5-dependent phosphorylation sites of amphiphysin 1 are implicated in the regulation of the membrane binding and endocytosis

Shuang Liang, Fan Yan Wei, Yu Mei Wu, Kenji Tanabe, Tadashi Abe, Yoshiya Oda, Yumi Yoshida, Hiroshi Yamada, Hideki Matsui, Kazuhito Tomizawa, Kohji Takei

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)1466-1476
Number of pages11
JournalJournal of Neurochemistry
Volume102
Issue number5
DOIs
Publication statusPublished - Sep 2007

Keywords

  • Amphiphysin
  • Clathrin-mediated endocytosis
  • Cyclin-dependent kinase 5
  • Endocytic protein
  • Liposome
  • Synaptic vesicle endocytosis

ASJC Scopus subject areas

  • Biochemistry
  • Cellular and Molecular Neuroscience

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