Structural insights into the tyrosine phosphorylation-mediated inhibition of SH3 domain-ligand interactions.

Src homology 3 (SH3) domains bind proline-rich linear motifs in eukaryotes. By mediating inter- and intramolecular interactions, they regulate the functions of many proteins involved in a wide variety of signal transduction pathways. Phosphorylation at different tyrosine residues in SH3 domains has been reported previously. In several cases, the functional consequences have also been investigated. However, a full understanding of the effects of tyrosine phosphorylation on the ligand interactions and cellular functions of SH3 domains requires detailed structural, atomic-resolution studies along with biochemical and biophysical analyses. Here, we present the first crystal structures of tyrosine-phosphorylated human SH3 domains derived from the Abelson-family kinases ABL1 and ABL2 at 1.6 and 1.4 Å resolutions, respectively. The structures revealed that simultaneous phosphorylation of Tyr89 and Tyr134 in ABL1 or the homologous residues Tyr116 and Tyr161 in ABL2 induces only minor structural perturbations. Instead, the phosphate groups sterically blocked the ligand-binding grooves, thereby strongly inhibiting the interaction with proline-rich peptide ligands. Although some crystal contact surfaces involving phosphotyrosines suggested the possibility of tyrosine phosphorylation-induced dimerization, we excluded this possibility by using small-angle X-ray scattering (SAXS), dynamic light scattering (DLS), and NMR relaxation analyses. Extensive analysis of relevant databases and literature revealed not only that the residues phosphorylated in our model systems are well-conserved in other human SH3 domains, but that the corresponding tyrosines are known phosphorylation sites in vivo in many cases. We conclude that tyrosine phosphorylation might be a mechanism involved in the regulation of the human SH3 interactome.

EGF Regulates the Interaction of Tks4 with Src through Its SH2 and SH3 Domains.

The nonreceptor tyrosine kinase Src is a central component of the epidermal growth factor (EGF) signaling pathway. Our group recently showed that the Frank-ter Haar syndrome protein Tks4 (tyrosine kinase substrate with four Src homology 3 domains) is also involved in EGF signaling. Here we demonstrate that Tks4 and Src bind directly to each other and elucidate the details of the molecular mechanism of this complex formation. Results of GST pull-down and fluorescence polarization assays show that both a proline-rich SH3 binding motif (PSRPLPDAP, residues 466-474) and an adjacent phosphotyrosine-containing SH2 binding motif (pYEEI, residues 508-511) in Tks4 are responsible for Src binding. These motifs interact with the SH3 and SH2 domains of Src, respectively, leading to a synergistic enhancement of binding strength and a highly stable, "bidentate"-type of interaction. In agreement with these results, we found that the association of Src with Tks4 is permanent and the complex lasts at least 3 h in living cells. We conclude that the interaction of Tks4 with Src may result in the long term stabilization of the kinase in its active conformation, leading to prolonged Src activity following EGF stimulation.

Dendritic spine morphology and memory formation depend on postsynaptic Caskin proteins.

CASK-interactive proteins, Caskin1 and Caskin2, are multidomain neuronal scaffold proteins. Recent data from Caskin1 knockout animals indicated only a mild role of Caskin1 in anxiety and pain perception. In this work, we show that deletion of both Caskins leads to severe deficits in novelty recognition and spatial memory. Ultrastructural analyses revealed a reduction in synaptic profiles and dendritic spine areas of CA1 hippocampal pyramidal neurons of double knockout mice. Loss of Caskin proteins impaired LTP induction in hippocampal slices, while miniature EPSCs in dissociated hippocampal cultures appeared to be unaffected. In cultured Caskin knockout hippocampal neurons, overexpressed Caskin1 was enriched in dendritic spine heads and increased the amount of mushroom-shaped dendritic spines. Chemically induced LTP (cLTP) mediated enlargement of spine heads was augmented in the knockout mice and was not influenced by Caskin1. Immunocytochemistry and immunoprecipitation confirmed that Shank2, a master scaffold of the postsynaptic density, and Caskin1 co-localized within the same complex. Phosphorylation of AMPA receptors was specifically altered by Caskin deficiency and was not elevated by cLTP treatment further. Taken together, our results prove a previously unnoticed postsynaptic role of Caskin scaffold proteins and indicate that Caskins influence learning abilities via regulating spine morphology and AMPA receptor localisation.

Significance of the Tks4 scaffold protein in bone tissue homeostasis.

The main driver of osteoporosis is an imbalance between bone resorption and formation. The pathogenesis of osteoporosis has also been connected to genetic alterations in key osteogenic factors and dysfunction of bone marrow mesenchymal stem/stromal cells (BM-MSCs). Tks4 (encoded by the Sh3pxd2b gene) is a scaffold protein involved in podosome organization. Homozygous mutational inactivation of Sh3pxd2b causes Frank-ter Haar syndrome (FTHS), a genetic disease that affects bone tissue as well as eye, ear, and heart functions. To date, the role of Tks4 in adult bone homeostasis has not been investigated. Therefore, the aim of this study was to analyze the facial and femoral bone phenotypes of Sh3pxd2b knock-out (KO) mice using micro-CT methods. In addition to the analysis of the Sh3pxd2b-KO mice, the bone microstructure of an FTHS patient was also examined. Macro-examination of skulls from Tks4-deficient mice revealed craniofacial malformations that were very similar to symptoms of the FTHS patient. The femurs of the Sh3pxd2b-KO mice had alterations in the trabecular system and showed signs of osteoporosis, and, similarly, the FTHS patient also showed increased trabecular separation/porosity. The expression levels of the Runx2 and osteocalcin bone formation markers were reduced in the bone and bone marrow of the Sh3pxd2b-KO femurs, respectively. Our recent study demonstrated that Sh3pxd2b-KO BM-MSCs have a reduced ability to differentiate into osteoblast lineage cells; therefore, we concluded that the Tks4 scaffold protein is important for osteoblast formation, and that it likely plays a role in bone cell homeostasis.

The scaffold protein Tks4 is required for the differentiation of mesenchymal stromal cells (MSCs) into adipogenic and osteogenic lineages.

The commitment steps of mesenchymal stromal cells (MSCs) to adipogenic and other lineages have been widely studied but not fully understood. Therefore, it is critical to understand which molecules contribute to the conversion of stem cells into differentiated cells. The scaffold protein Tks4 plays a role in podosome formation, EGFR signaling and ROS production. Dysfunction of Tks4 causes a hereditary disease called Frank-ter Haar syndrome with a variety of defects concerning certain mesenchymal tissues (bone, fat and cartilage) throughout embryogenic and postnatal development. In this study, we aimed to analyze how the mutation of Tks4 affects the differentiation potential of multipotent bone marrow MSCs (BM-MSCs). We generated a Tks4 knock-out mouse strain on C57Bl/6 background, and characterized BM-MSCs isolated from wild type and Tks4-/- mice to evaluate their differentiation. Tks4-/- BM-MSCs had reduced ability to differentiate into osteogenic and adipogenic lineages compared to wild type. Studying the expression profile of a panel of lipid-regulated genes during adipogenic induction revealed that the expression of adipogenic transcription factors, genes responsible for lipid droplet formation, sterol and fatty acid metabolism was delayed or reduced in Tks4-/- BM-MSCs. Taken together, these results establish a novel function for Tks4 in the regulation of MSC differentiation.