Direct binding of plectin to Fer kinase and negative regulation of its catalytic activity

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Abstract

Plectin is a cyoskeletal linker protein that protects tissues against mechanical stress. We report here that the N-terminal domain of the nonreceptor tyrosine kinase Fer interacts with N-terminal sequences of plectin. Recombinant protein encoded by exon 12–24 of rat plectin bound directly to amino acid 1–329 of murine Fer. Using an antiserum prepared to a recombinant N-terminal fragment of Fer kinase, plectin was coimmunoprecipitated with Fer from cell lysates of cultured mouse fibroblasts. Plectin was shown to partially colocalize with Fer in these cells. Upon transfection of full length Fer cDNA into plectin-negative mouse fibroblasts, hyperphosphorylation of Fer was observed; hyperphosphorylation was strongly reduced when N-terminal Fer deletion mutants were transfected. Immunocomplex kinase assays showed that the activity of Fer kinase transfected into plectin-negative fibroblasts was increased compared to that transfected into wild type cells. We conclude that Fer interacts with plectin and that this interaction may serve to negatively regulate Fer's activity.

Section snippets

Materials and methods

Preparation of DNA constructs. Plasmid pMZ4, encoding histidine-tagged rat plectin sequences (exon 12–24), has been described previously [19]. Plasmid pPL2, encoding full length murine Fer with an N-terminal histidine tag, was constructed by amplifying a 500 bp fragment of the plasmid pECE-mufer (provided by Dr. A. J. Pawson) using as upper primer 5CTTACAGATCTGGATTTGGGAGTGACCTGAAG3 and 5TGTTTCCTTCCCTTTCGCTAAGGC3 as lower primer. The product was digested using BglII and EcoRI and cloned into

The N-terminal domain of Fer interacts directly with plectin

Screening of a mouse cDNA library in the yeast two-hybrid system using the N-terminal domain of murine Fer as bait originally uncovered a fragment of plectin encoded by exons 14–19 as a potential interaction partner of the kinase. To confirm this observation, corresponding fragments were expressed as fusion proteins containing either maltose-binding protein (MBP-Fer) or a his-tag (his-plectin) and assayed for binding in vitro. MBP-Fer containing the first 329 N-terminal amino acids of the Fer

Discussion

Protein–protein interactions between tyrosine kinases and their targets, mediated by specialized protein modules, determine the pathways along which signals are transduced and therefore how their signaling networks are organized. Apart from direct interactions between signaling proteins, interactions between signaling proteins and scaffolding proteins also determine to which targets, and with what signal strength and speed, signals are transduced. In this study, we show that the N-terminal

Acknowledgements

The authors thank Dr. Anthony J. Pawson for the donation of reagents and Mag. Branislav Nikolic for assistance and helpful discussions. This research work was supported by a Marie Curie Fellowship of the European Union programme Training and Mobility of Researchers (TMR) under contract number ERBFMBICT972397 (to P.L.) and a grant from the Austrian Science Research Fund (P14520).

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    Abbreviations: SH2, Src homology 2; MBP, maltose-binding protein; EGFP, enhanced green fluorescent protein; tTA, Tet-responsive transactivator; mAb, monoclonal antibody.

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