Binding of Insulin Receptor Substrate 1
PH Domain to Protein Kinase C
JI Shao-Ping,YAO Li-Bo,BAI Yu-Jie,WANG Ji-Cun,LIU Xin-Ping
and SU Cheng-Zhi
( Department of Biochemistry and Molecular Biology,Fourth Military Medical University,Xi’an
710032,China )
Abstract To screen for novel ligands of
insulin receptor substrate 1(IRS-1)PH domains,to investigate the role of the PH
domains in signal transduction processes and to examine association of the PH
domain with protein kinase C(PKC),rat liver was employed to clone the gene
encoding for the PH domains.Total RNAs were isolated and purified from fresh
liver and mRNAs were reversely transcribed into cDNAs.After PCR the fragments
of the DNA were cloned into vector pUC19.The sequence of the fusion gene was
confirmed by sequencing,and the gene was correctly expressed in E.coli
as fusion protein with glutathione S― transferase(GST).The fusion protein was
purified by glutathione agarose beads,then was incubated with the lysate of
Jurkat cells.After SDS-PAGE,the proteins were transferred to PVDF membrane,and
an anti-PKC antibody was used to detect binding between the PH domain with
PKC.The sequence of the gene encoding for the PH domains was confirmed to be
correct,and the PH domain was successfully expressed in E.coli JM 109
in soluble form.Western blots confirmed the binding of the PH domain with PKC in
vitro.In conclusion,purified IRS-1 PH domain GST fusion protein was
obtained and its biological activity was confirmed.
Key Words Signal transduction；IRS-1；PH domain；clone；expression
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