Liprin phosphorylation regulates binding to LAR: evidence for liprin autophosphorylation.
The LAR transmembrane tyrosine phosphatase associates with liprin-alpha proteins and colocalizes with liprin-alpha1 at focal adhesions. LAR has been implicated in axon guidance, and liprins are involved in synapse formation and synapse protein trafficking. Several liprin mutants have weaker binding to LAR as assessed by yeast interaction trap assays, and the extents of in vitro and in vivo phosphorylation of these mutants were reduced relative to that of wild-type liprin-alpha1. Treatment of liprin-alpha1 with calf intestinal phosphatase weakened its interaction with the recombinant GST-LAR protein. A liprin LH region mutant that inhibited liprin phosphorylation did not bind to LAR as assessed by coprecipitation studies. Endogenous LAR was shown to bind phosphorylated liprin-alpha1 from MDA-486 cells labeled in vivo with [32P]orthophosphate. In further characterizing the phosphorylation of liprin, we found immunoprecipitates of liprin-alpha1 expressed in COS-7 cells to incorporate phosphate after washes of up to 4 M NaCl. Additionally, purified liprin-alpha1 derived from Sf-9 insect cells retained the ability to incorporate phosphate in in vitro phosphorylation assays, and a liprin-alpha1 truncation mutant incorporated phosphate after denaturation 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 LH region 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.
Serra-Pagès
Carles
C
Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, Massachusetts 02115, USA.
Streuli
Michel
M
Medley
Quintus G
QG
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D006801:Humans; D008565:Membrane Proteins / Q000096:biosynthesis; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism*; D054623:Receptor-Like Protein Tyrosine Phosphatases, Class 2; D011956:Receptors, Cell Surface / Q000378:metabolism*; D016415:Sequence Alignment
D048868:Adaptor Proteins, Signal Transducing; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; C094398:PPFIA1 protein, human; C470384:PPFIA2 protein, human; D010750:Phosphoproteins; D011956:Receptors, Cell Surface; C516772:PTPRF protein, human; D017027:Protein Tyrosine Phosphatases; D054623:Receptor-Like Protein Tyrosine Phosphatases, Class 2