Mice devoid of fer protein-tyrosine kinase activity are viable and fertile but display reduced cortactin phosphorylation

Abstract

The ubiquitous Fer protein-tyrosine kinase has been proposed to regulate diverse processes such as cell growth, cell adhesion, and neurite outgrowth. To gain insight into the biological function of Fer, we have targeted the fer locus with a kinase-inactivating missense mutation (fer(D743R)). Mice homozygous for this mutation develop normally, have no overt phenotypic differences from wild-type mice, and are fertile. Since these mice lack both Fer and the testis-specific FerT kinase activities, these proteins are clearly not essential for development and survival. No differences were observed in overall cellularity of bone marrow, spleen, or thymus in the absence of Fer activity. While most platelet-derived growth factor (PDGF)-induced tyrosine phosphorylation was unchanged in fer(D743R) homozygous embryonic fibroblasts, cortactin phosphorylation was reduced. However, Fer kinase activity was not required for PDGF-induced Stat3, p120(ctn), or epidermal growth factor (EGF)-induced beta-catenin phosphorylation. Also, no defects were observed in changes to the actin cytoskeleton, adherens junctions, or focal adhesions in PDGF- or EGF-stimulated fer(D743R) homozygous embryonic fibroblasts. Therefore, Fer likely serves a redundant role in regulating cell growth, cell adhesion, retinal development, and spermatogenesis but is required for efficient phosphorylation of cortactin.

FIG. 1

Domain structure of Fer and targeting strategy of fer locus. (A) Fer consists of three CC motifs, a central SH2 domain, and a kinase domain. The catalytic subdomains are shown, with subdomain IX encoded by exon 19 contained within genomic clone 3B. The targeting vector contains a 6-kbp long arm of homology, followed by the PGK-neo cassette, a 0.9-kbp short arm of homology, and a PGK-tk cassette. The D743R mutation was generated within exon 19, which also introduces an NruI (N) site. A schematic of the targeted allele following homologous recombination is shown, with positions of PCR primers and Southern blot probe indicated. Positions of XbaI (X), XhoI (Xh), NheI (Nh), EcoRI (E), and SacI (S) sites are shown. Sites in parentheses were destroyed. (B) Southern blot analysis of SacI-digested genomic DNA from animals that were wild type (+/+), heterozygous (+/−), or homozygous (−/−) for fer^D743R. Hybridization with a probe located 3′ to the sequence used for the short arm of homology reveals bands of 4.0 kbp for the wild-type allele and 5.8 kbp for the fer^D743R allele. (C) Genotyping PCR analysis of genomic DNA from +/+, +/−, or −/− animals. The resulting 871-bp fragment is resistant to NruI digestion in wild-type samples, yields additional 605- and 266-bp bands in heterozygous samples, and yields only 605- and 266-bp fragments in homozygous samples.

FIG. 2

Analysis of Fer expression and kinase activity in fer^D743R mice. (A) Liver homogenates from wild-type (+/+), heterozygous (+/−), and homozygous (−/−) fer^D743R mice (strain 153) were subjected to immunoprecipitation (IP) with Fer antisera, followed by in vitro kinase reactions. The position of Fer is indicated on the left. (B) Testis homogenates from +/+, +/−, or −/− fer^D743R male mice were subjected to immunoprecipitation with Fps/Fer antisera (raised against the SH2 domain of Fps but cross-reactive with Fer and FerT), followed by in vitro kinase reactions. The positions of Fer, FerT, and the IgG heavy chain are indicated on the left. Cross-reacting bands in the soluble cell lysate, and a putative partial degradation product in the kinase assay, are indicated with asterisks.

FIG. 3

Analysis of major PDGF signaling pathways in fer^D743R embryonic fibroblasts. Wild-type (+/+), heterozygous (+/−), and homozygous (−/−) fer^D743R cells were starved and stimulated with PDGF-BB for 5 min. Soluble cell lysates were separated by SDS-PAGE and blotted for tyrosine-phosphorylated proteins (pY), Fer, phospho-Akt (pAkt), Akt, phospho-Stat3 (pStat3), Stat3, phospho-Erk1/2 (pErk1/2), and Erk1/2. Relative molecular weights are indicated in thousands on the left.

FIG. 4

Impaired cortactin but not p120^ctn phosphorylation in fer^D743R embryonic fibroblasts. Wild-type (+/+), heterozygous (+/−), and homozygous (−/−) fer^D743R cells were starved and stimulated with PDGF-BB for 5 min. Lysates were subjected to immunoprecipitation (IP) with Fer, p120^ctn, and cortactin antibodies. Soluble cell lysates and immunoprecipitates were resolved by SDS-PAGE and blotted for tyrosine-phosphorylated proteins (pY), Fer, p120^ctn, or cortactin. Relative molecular weight markers are indicated in thousands on the left.

FIG. 5

EGF-stimulated β-catenin phosphorylation in the absence of Fer activity. Wild-type (+/+) and homozygous (−/−) fer^D743R cells were starved and stimulated with EGF for 5 min. Lysates were subjected to immunoprecipitation (IP) with Fer and β-catenin-specific antibodies. Soluble cell lysates and immune complexes were resolved by SDS-PAGE and blotted for tyrosine-phosphorylated proteins (pY), Fer, and β-catenin. Relative molecular weight markers are indicated in thousands on the left.

FIG. 6

Localization of the actin cytoskeleton and of actin-, cadherin-, or focal adhesion-associated proteins in fer^D743R embryonic fibroblasts. Wild-type (+/+) and homozygous (−/−) fer^D743R cells were plated on gelatinized coverslips, starved of growth factors, and stimulated with either PDGF (A) or EGF (B). The proteins denoted on the left were detected with monoclonal antibodies and Alexa Fluor 568 goat anti-mouse secondary (indicated by red color). Actin cytoskeleton was visualized by staining with FITC-phalloidin (indicated by green color). Yellow denotes colocalization of protein and actin. Representative images are shown following confocal microscopy.