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ABSTRACT: 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.
Biochemistry 01/2006; 44(48):15715-24. · 3.42 Impact Factor
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ABSTRACT: The Trio guanine nucleotide exchange factor functions in neural development in Caenorhabditis elegans and Drosophila and in the development of neural tissues and skeletal muscle in mouse. The association of Trio with the Lar tyrosine phosphatase led us to study the role of tyrosine phosphorylation in Trio function using focal adhesion kinase (FAK). The Lar-interacting domain of Trio is constitutively tyrosine-phosphorylated when expressed in COS-7 cells and was highly phosphorylated when it was co-transfected with FAK. Co-precipitation studies indicated that Trio binds to the FAK amino-terminal domain and to the FAK kinase domain via its SH3 and kinase domains, respectively. Tyrosine-phosphorylated FAK and Trio were present mainly in the detergent-insoluble fraction of cell lysates, and co-expression of Trio and FAK resulted in increased amounts of Trio present in the detergent-insoluble fraction. Immunofluorescence of cells co-transfected with FAK and Trio revealed significant co-localization of the proteins at the cell periphery, indicating that they form a stable complex in vivo. A FAK phosphorylation site, tyrosine residue 2737, was identified in subdomain I of the Trio kinase domain. Additionally, in vitro phosphorylation assays and in vivo co-expression studies indicated that Trio enhances FAK kinase activity. These results suggest Trio may be involved in the regulation of focal adhesion dynamics in addition to effecting changes in the actin cytoskeleton through the activation of Rho family GTPases.
Journal of Biological Chemistry 05/2003; 278(15):13265-70. · 4.77 Impact Factor
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ABSTRACT: Interaction with the multi-PDZ protein GRIP is required for the synaptic targeting of AMPA receptors, but the underlying mechanism is unknown. We show that GRIP binds to the liprin-alpha/SYD2 family of proteins that interact with LAR receptor protein tyrosine phosphatases (LAR-RPTPs) and that are implicated in presynaptic development. In neurons, liprin-alpha and LAR-RPTP are enriched at synapses and coimmunoprecipitate with GRIP and AMPA receptors. Dominant-negative constructs that interfere with the GRIP-liprin interaction disrupt the surface expression and dendritic clustering of AMPA receptors in cultured neurons. Thus, by mediating the targeting of liprin/GRIP-associated proteins, liprin-alpha is important for postsynaptic as well as presynaptic maturation.
Neuron 04/2002; 34(1):39-52. · 14.74 Impact Factor
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ABSTRACT: Trio is a complex protein containing two guanine nucleotide exchange factor domains each with associated pleckstrin homology
domains, a serine/threonine kinase domain, two SH3 domains, an immunoglobulin-like domain, and spectrin-like repeats. Trio
was originally identified as a LAR tyrosine phosphatase-binding protein and is involved in actin remodeling, cell migration,
and cell growth. Herein we provide evidence that Trio not only activates RhoA but is also a RhoA target. The RhoA-binding
site was mapped to the Trio immunoglobulin-like domain. RhoA isoprenylation is necessary for the RhoA-Trio interaction, because
mutation of the RhoA carboxyl-terminal cysteine residue blocked binding. The existence of an intramolecular functional link
between RhoA activation and RhoA binding is suggested by the finding that Trio exchange activity enhanced RhoA binding to
Trio. Furthermore, immunofluorescence studies of HeLa cells showed that although ectopically expressed Trio was evenly distributed
within the cell, co-expression of Trio with RhoA resulted in relocalization of Trio into punctate structures. Relocalization
was not observed with Trio constructs lacking the immunoglobulin-like domain, indicating that RhoA acts to regulate Trio localization
via binding to the immunoglobulin-like domain. We propose that Trio-mediated RhoA activation and subsequent RhoA-mediated
relocalization of Trio functions to modulate and coordinate Trio signaling.
Journal of Biological Chemistry 11/2000; 275(46):36116-36123. · 4.77 Impact Factor
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ABSTRACT: Protein-tyrosine phosphatases (PTPases) play an essential role in the regulation of cell activation, proliferation, and differentiation.
A major subfamily of these enzymes is the transmembrane-type PTPases that contain extracellular regions comprised of Ig-like
and fibronectin type III (FN-III)-like domains. Characterization of the human transmembrane PTPase δ (HPTPδ) revealed the
existence of multiple HPTPδ isoforms that vary in their extracellular regions. The full-length HPTPδ isoform has an extracellular
region containing three Ig-like and eight FN-III-like domains connected via a transmembrane peptide to an intracellular region
with two PTPase domains, whereas another isoform lacks four of the eight FN-III like domains. Furthermore, other HPTPδ isoforms
exist that lack 9 amino acids within the second Ig-like domain and 4 amino acids at the junction of the second and third Ig-like
domains or 9 amino acids within the fifth FN-III-like domain. Reverse transcription polymerase chain reaction analysis demonstrated
that HPTPδ isoforms lacking these short peptides are expressed in kidney, whereas isoforms containing these peptides are expressed
in the brain. Analysis of HPTPδ biosynthesis demonstrated that HPTPδ is expressed as a complex of two noncovalently associated
subunits derived from a proprotein and that the HPTPδ ectodomain is shed from the cell surface. Mutational analysis of the
HPTPδ proprotein cleavage site revealed the existence of two or three functional and overlapping furin-like endoprotease cleavage
sites.
Journal of Biological Chemistry 03/1995; 270(12):6722-6728. · 4.77 Impact Factor
