Liprin Phosphorylation Regulates Binding to LAR: Evidence for Liprin Autophosphorylation
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- 作者：Carles Serra-Pagè ; s ; Michel Streuli ; and Quintus G. Medley
- 刊名：Biochemistry
- 出版年：2005
- 出版时间：December 6, 2005
- 年：2005
- 卷：44
- 期：48
- 页码：15715 - 15724
- 全文大小：1018K
- 年卷期：v.44,no.48(December 6, 2005)
- ISSN：1520-4995
文摘
The LAR transmembrane tyrosine phosphatase associates with liprin-
proteins and colocalizeswith liprin-1 at focal adhesions. LAR has been implicated in axon guidance, and liprins are involved insynapse formation and synapse protein trafficking. Several liprin mutants have weaker binding to LARas assessed by yeast interaction trap assays, and the extents of in vitro and in vivo phosphorylation ofthese mutants were reduced relative to that of wild-type liprin-1. Treatment of liprin-1 with calf intestinalphosphatase weakened its interaction with the recombinant GST-LAR protein. A liprin LH region mutantthat inhibited liprin phosphorylation did not bind to LAR as assessed by coprecipitation studies. EndogenousLAR was shown to bind phosphorylated liprin-1 from MDA-486 cells labeled in vivo with[32P]orthophosphate. In further characterizing the phosphorylation of liprin, we found immunoprecipitatesof liprin-1 expressed in COS-7 cells to incorporate phosphate after washes of up to 4 M NaCl.Additionally, purified liprin-1 derived from Sf-9 insect cells retained the ability to incorporate phosphatein in vitro phosphorylation assays, and a liprin-1 truncation mutant incorporated phosphate afterdenaturation and/or renaturation in SDS gels. Finally, binding assays showed that liprin binds to ATP-agarose and that the interaction is challenged by free ATP, but not by free GTP. Moreover, liprin LHregion mutations that inhibit liprin phosphorylation stabilized the association of liprin with ATP-agarose.Taken together, our results suggest that liprin autophosphorylation regulates its association with LAR.
proteins and colocalizeswith liprin-1 at focal adhesions. LAR has been implicated in axon guidance, and liprins are involved insynapse formation and synapse protein trafficking. Several liprin mutants have weaker binding to LARas assessed by yeast interaction trap assays, and the extents of in vitro and in vivo phosphorylation ofthese mutants were reduced relative to that of wild-type liprin-1. Treatment of liprin-1 with calf intestinalphosphatase weakened its interaction with the recombinant GST-LAR protein. A liprin LH region mutantthat inhibited liprin phosphorylation did not bind to LAR as assessed by coprecipitation studies. EndogenousLAR was shown to bind phosphorylated liprin-1 from MDA-486 cells labeled in vivo with[32P]orthophosphate. In further characterizing the phosphorylation of liprin, we found immunoprecipitatesof liprin-1 expressed in COS-7 cells to incorporate phosphate after washes of up to 4 M NaCl.Additionally, purified liprin-1 derived from Sf-9 insect cells retained the ability to incorporate phosphatein in vitro phosphorylation assays, and a liprin-1 truncation mutant incorporated phosphate afterdenaturation and/or renaturation in SDS gels. Finally, binding assays showed that liprin binds to ATP-agarose and that the interaction is challenged by free ATP, but not by free GTP. Moreover, liprin LHregion mutations that inhibit liprin phosphorylation stabilized the association of liprin with ATP-agarose.Taken together, our results suggest that liprin autophosphorylation regulates its association with LAR.