SUNDAY, DECEMBER 4- Late Abstracts 1 - Molecular Biology of the ...

SUNDAY
integrin to trigger the aggregation of lipid raft. The duration of lipid-raft aggregation was
extended from 10 minutes (induced by ligand-coated micro-bead) to 2 hours (induced by ligandcoated
Qdot), and finally the aggregate reached a diameter around 5 micrometers. In our
studies, RGD-coated Qdot bound to a small number of integrin proteins may provide an
effective means to observe the early events of lipid raft aggregation in a living cell.
2003
Molecular Mechanism of F-BAR Protein Pacsin2 in Caveolae Biogenesis.
Y. Senju 1 , S. Suetsugu 1 ; 1 Institute of Molecular and Cellular Biosciences, The University of
Tokyo, Tokyo, Japan
Protein kinase C (PKC) and casein kinase substrate in neurons 2 (Pacsin2) belongs to the
EFC/F-BAR domain protein family and deforms membrane. The N-terminal F-BAR domain of
pacsin2 forms a crescent-shaped dimer with a positively charged surface. The C-terminal SH3
domain associates with dynamin2, a GTPase implicated in vesicle endocytosis. Therefore,
pacsin2 is involved in membrane invaginations including caveolae (Senju et al., 2011). Previous
study has demonstrated that the cells overexpressing pacsin2 showed membrane invaginations.
However, the number of membrane invagination was larger in the cells overexpressing pacsin2
F-BAR domain than in the cells overexpressing pacsin2 full length, which was interpreted as
autoinhibition of pacsin2 resulted from the intramolecular interaction between F-BAR domain
and SH3 domain. Because pacsin2 has been identified as a PKC substrate, we investigated the
autoinhibition mechanism based on the phosphorylation of pacsin2 by PKC. First, we performed
an in vitro kinase assay, and confirmed that PKC actually phosphorylated pacsin2. Then, we
generated two different pacsin2 mutants by substitution with either alanine or glutamic acid, and
identified the phosphorylation site in pacsin2 by in vitro kinase assay. Next, we generated a
phospho-specific antibody, and confirmed that endogenous pacsin2 was phosphorylated after
the PKC activator PMA treatment using immunofluorescence microscopy and western blotting
analysis. The number of membrane invaginations induced by phospho-mimic mutant of pacsin2
was lesser than that induced by wild-type pacsin2. After the PMA treatment, the number of
membrane invagination was decreased in the cells expressing wild-type pacsin2, but there was
no significant effect in the cells expressing either phospho-mimic, phospho-deficient mutant, or
F-BAR domain of pacsin2. Therefore, these data suggest that phosphorylated pacsin2 prefers
an open protein conformation that permits interaction with its partners such as dynamin2. The
interaction of pacsin2 with dynamin2 probably caused dynmin2-induced scission of membrane
invaginations, which in turn decreased the number of pacsin2-induced membrane invaginations.
Further experimentation will be required to test this hypothesis and reveal the physiological role
of pacsin2 in caveolae biogenesis.
2004
Identification and characterization of atypical FYVE Domains for specific interaction with
phospholipids.
J-E. Gil 1 , I-S. Kim 2 , B. Ku 1 , W. Park 1 , B-H. Oh 1 , S. Ryu 2 , W. Heo 1 ; 1 Korea Advanced Institute of
Science and Technology, Daejeon, Korea, 2 Pohang University of Science and Technology,
Korea
Interactions of lipid binding domains with intracellular lipids play a significant role in regulation of
numerous signaling and cellular mechanisms. To characterize interactions of lipid binding
domains with phospholipids, five classes of lipid binding domains were screened by fluorescent
imaging and classified based on their subcellular localizations. Six of over one hundred kinds of
lipid binding domains were localized in the plasma membrane where a number of signaling
cascades are generally initiated. Interestingly, we found FYVE domain of ZFYVE27 which is