Identification of p115 as a PLCgamma1-binding protein and the role of Src homology domains of PLCgamma1 in the vesicular transport.
In order to gain further insight into the function(s) of PLCgamma1, we tried to identify the binding partners that can interact with the SH223 domains of PLCgamma1. Immunoscreening was performed with the purified antisera that are specific to SH223-binding proteins. Several immunoreactive clones were identified as the putative binding proteins and one of them was identified as p115. p115 was reported to be required for transcytotic fusion and subsequent binding of the vesicles to the target membrane. The interaction between PLCgamma1 and p115 was specific to carboxyl-terminal SH2 domain and SH3 domain of PLCgamma1, and also confirmed by biochemical approaches such as co-immunoprecipitation, pull-down assay, and glycerol gradient fractionation. To further characterize the role of SH domains of PLCgamma1 in the vesicle transport pathway, secreted form of alkaline phosphatase (SEAP) reporter assay was carried out. When the SH2 and/or SH3 domains of PLCgamma1 were deleted, the secretion of SEAP was significantly reduced. These findings indicate that the SH2 and SH3 domains of PLCgamma1 may play a role(s) in the process of the vesicle transport via interaction with other vesicle-associated proteins such as p115.
Han
Seung Jin
SJ
School of Biological Sciences, Seoul National University, Seoul, Republic of Korea.
Lee
Jung Hyun
JH
Kim
Chan Gil
CG
Hong
Seung Hwan
SH
D000818:Animals; D001692:Biological Transport / Q000502:physiology; D001923:Brain Chemistry; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D000077614:Golgi Matrix Proteins; D006801:Humans; D015151:Immunoblotting; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016716:PC12 Cells; D051966:Phospholipase C gamma; D011233:Precipitin Tests; D011485:Protein Binding / Q000502:physiology; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity / Q000502:physiology; D014162:Transfection; D010738:Type C Phospholipases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D033921:Vesicular Transport Proteins; D018909:src Homology Domains / Q000502:physiology*
D002352:Carrier Proteins; D000077614:Golgi Matrix Proteins; D007527:Isoenzymes; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D033921:Vesicular Transport Proteins; C091580:vesicular transport factor p115; D010738:Type C Phospholipases; D051966:Phospholipase C gamma