Contribution of Noncovalent Recognition and Reactivity to the Optimization of Covalent Inhibitors: A Case Study on KRasG12C.

Covalent drugs might bear electrophiles to chemically modify their targets and have the potential to target previously undruggable proteins with high potency. Covalent binding of drug-size molecules includes a noncovalent recognition provided by secondary interactions and a chemical reaction leading to covalent complex formation. Optimization of their covalent mechanism of action should involve both types of interactions. Noncovalent and covalent binding steps can be characterized by an equilibrium dissociation constant (KI) and a reaction rate constant (kinact), respectively, and they are affected by both the warhead and the scaffold of the ligand. The relative contribution of these two steps was investigated on a prototypic drug target KRASG12C, an oncogenic mutant of KRAS. We used a synthetically more accessible nonchiral core derived from ARS-1620 that was equipped with four different warheads and a previously described KRAS-specific basic side chain. Combining these structural changes, we have synthesized novel covalent KRASG12C inhibitors and tested their binding and biological effect on KRASG12C by various biophysical and biochemical assays. These data allowed us to dissect the effect of scaffold and warhead on the noncovalent and covalent binding event. Our results revealed that the atropisomeric core of ARS-1620 is not indispensable for KRASG12C inhibition, the basic side chain has little effect on either binding step, and warheads affect the covalent reactivity but not the noncovalent binding. This type of analysis helps identify structural determinants of efficient covalent inhibition and may find use in the design of covalent agents.

Unveiling Epithelial Plasticity Regulation in Lung Cancer: Exploring the Crosstalk Among Tks4 Scaffold Protein Partners.

The epithelial to mesenchymal transition (EMT) represents a hallmark event in the evolution of lung cancer. This work aims to study a recently described EMT-regulating protein,Tks4, and to explore its potential as a prognostic biomarker in non-small cell lung cancer (NSCLC). In this study, we used CRISPR/Cas9 method to knockout Tks4 to study its functional roles in invadopodia formation, migration, and regulation of EMT marker expressions and we identified Tks4-interacting proteins. Tks4-KO A549 cells exhibited an EMT-like phenotype characterized by elongated morphology and increased expression of EMT markers. Furthermore, analyses of a large-scale lung cancer database and a patient-derived tissue array data revealed that the Tks4 mRNA level was decreased in more aggressive lung cancer stages. To understand the regulatory role of Tks4 in lung cancer, we performed a Tks4-interactome analysis via Tks4 immunoprecipitation-mass spectroscopy on four different cell lines and identified CAPZA1 as a novel Tks4 partner protein. Thus, we propose that the absence of Tks4 leads to disruption of a connectome of multiple proteins and that the resulting undocking and likely mislocalization of signaling molecules impairs actin cytoskeleton rearrangement and activates EMT-like cell fate switches, both of which likely influence disease severity.

Studying the Association of TKS4 and CD2AP Scaffold Proteins and Their Implications in the Partial Epithelial-Mesenchymal Transition (EMT) Process.

Colon cancer is a leading cause of death worldwide. Identification of new molecular factors governing the invasiveness of colon cancer holds promise in developing screening and targeted therapeutic methods. The Tyrosine Kinase Substrate with four SH3 domains (TKS4) and the CD2-associated protein (CD2AP) have previously been linked to dynamic actin assembly related processes and cancer cell migration, although their co-instructive role during tumor formation remained unknown. Therefore, this study was designed to investigate the TKS4-CD2AP interaction and study the interdependent effect of TKS4/CD2AP on oncogenic events. We identified CD2AP as a novel TKS4 interacting partner via co-immunoprecipitation-mass spectrometry methods. The interaction was validated via Western blot (WB), immunocytochemistry (ICC) and proximity ligation assay (PLA). The binding motif of CD2AP was explored via peptide microarray. To uncover the possible cooperative effects of TKS4 and CD2AP in cell movement and in epithelial-mesenchymal transition (EMT), we performed gene silencing and overexpressing experiments. Our results showed that TKS4 and CD2AP form a scaffolding protein complex and that they can regulate migration and EMT-related pathways in HCT116 colon cancer cells. This is the first study demonstrating the TKS4-CD2AP protein-protein interaction in vitro, their co-localization in intact cells, and their potential interdependent effects on partial-EMT in colon cancer.

Absence of Scaffold Protein Tks4 Disrupts Several Signaling Pathways in Colon Cancer Cells.

Tks4 is a large scaffold protein in the EGFR signal transduction pathway that is involved in several cellular processes, such as cellular motility, reactive oxygen species-dependent processes, and embryonic development. It is also implicated in a rare developmental disorder, Frank-ter Haar syndrome. Loss of Tks4 resulted in the induction of an EMT-like process, with increased motility and overexpression of EMT markers in colorectal carcinoma cells. In this work, we explored the broader effects of deletion of Tks4 on the gene expression pattern of HCT116 colorectal carcinoma cells by transcriptome sequencing of wild-type and Tks4 knockout (KO) cells. We identified several protein coding genes with altered mRNA levels in the Tks4 KO cell line, as well as a set of long non-coding RNAs, and confirmed these changes with quantitative PCR on a selected set of genes. Our results show a significant perturbation of gene expression upon the deletion of Tks4, suggesting the involvement of different signal transduction pathways over the well-known EGFR signaling.

[Farnesyltransferase inhibitors have antitumoral effects in mutant KRAS containing cancer cells in preclinical models]. / Farnezil-transzferáz-inhibitorok (FTI) szinergista módon potencírozzák a mutáns KRAS inhibitorai daganatellenes hatásait preklinikai modellekben.

In silico studies raised the possibility that farnesyltransferase inhibitors (FTIs) may have antitumoral effects on KRAS mutant cancer cells. Accordingly, we have tested FTIs (tipifarnib and lonafarnib) in G12C mutant human cancer cell lines in vitro and in vivo. We have discovered that the combination of the two drugs has a synergistic antitumoral effect. Next, we have tested FTIs on G12D mutant human cancer cell lines and found that the combination has antitumoral effect in various preclinical cancer models. At last, we have also tested FTIs on G12V mutant human cancer cells and again we have detected antitumoral effects. We suggest that FTIs may have clinical relevance outside the HRAS mutant cancers.

A Novel Cell-Based Model for a Rare Disease: The Tks4-KO Human Embryonic Stem Cell Line as a Frank-Ter Haar Syndrome Model System.

Tyrosine kinase substrate with four SH3 domains (Tks4) scaffold protein plays roles in cell migration and podosome formation and regulates systemic mechanisms such as adult bone homeostasis and adipogenesis. Mutations in the Tks4 gene (SH3PXD2b) cause a rare developmental disorder called Frank-Ter Haar syndrome (FTHS), which leads to heart abnormalities, bone tissue defects, and reduced adiposity. We aimed to produce a human stem cell-based in vitro FTHS model system to study the effects of the loss of the Tks4 protein in different cell lineages and the accompanying effects on the cell signalome. To this end, we used CRISPR/Cas9 (clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR associated (Cas9)) to knock out the SH3PXD2b gene in the HUES9 human embryonic stem cell line (hESC), and we obtained stable homo- and heterozygous knock out clones for use in studying the potential regulatory roles of Tks4 protein in embryonic stem cell biology. Based on pluripotency marker measurements and spontaneous differentiation capacity assays, we concluded that the newly generated Tks4-KO HUES9 cells retained their embryonic stem cell characteristics. We propose that the Tks4-KO HUES9 cells could serve as a tool for further cell differentiation studies to investigate the involvement of Tks4 in the complex disorder FTHS. Moreover, we successfully differentiated all of the clones into mesenchymal stem cells (MSCs). The derived MSC cultures showed mesenchymal morphology and expressed MSC markers, although the expression levels of mesodermal and osteogenic marker genes were reduced, and several EMT (epithelial mesenchymal transition)-related features were altered in the Tks4-KO MSCs. Our results suggest that the loss of Tks4 leads to FTHS by altering cell lineage differentiation and cell maturation processes, rather than by regulating embryonic stem cell potential.

Characterization of the Intramolecular Interactions and Regulatory Mechanisms of the Scaffold Protein Tks4.

The scaffold protein Tks4 is a member of the p47phox-related organizer superfamily. It plays a key role in cell motility by being essential for the formation of podosomes and invadopodia. In addition, Tks4 is involved in the epidermal growth factor (EGF) signaling pathway, in which EGF induces the translocation of Tks4 from the cytoplasm to the plasma membrane. The evolutionarily-related protein p47phox and Tks4 share many similarities in their N-terminal region: a phosphoinositide-binding PX domain is followed by two SH3 domains (so called "tandem SH3") and a proline-rich region (PRR). In p47phox, the PRR is followed by a relatively short, disordered C-terminal tail region containing multiple phosphorylation sites. These play a key role in the regulation of the protein. In Tks4, the PRR is followed by a third and a fourth SH3 domain connected by a long (~420 residues) unstructured region. In p47phox, the tandem SH3 domain binds the PRR while the first SH3 domain interacts with the PX domain, thereby preventing its binding to the membrane. Based on the conserved structural features of p47phox and Tks4 and the fact that an intramolecular interaction between the third SH3 and the PX domains of Tks4 has already been reported, we hypothesized that Tks4 is similarly regulated by autoinhibition. In this study, we showed, via fluorescence-based titrations, MST, ITC, and SAXS measurements, that the tandem SH3 domain of Tks4 binds the PRR and that the PX domain interacts with the third SH3 domain. We also investigated a phosphomimicking Thr-to-Glu point mutation in the PRR as a possible regulator of intramolecular interactions. Phosphatidylinositol-3-phosphate (PtdIns(3)P) was identified as the main binding partner of the PX domain via lipid-binding assays. In truncated Tks4 fragments, the presence of the tandem SH3, together with the PRR, reduced PtdIns(3)P binding, while the presence of the third SH3 domain led to complete inhibition.

Cellular Chaperone Function of Intrinsically Disordered Dehydrin ERD14.

Disordered plant chaperones play key roles in helping plants survive in harsh conditions, and they are indispensable for seeds to remain viable. Aside from well-known and thoroughly characterized globular chaperone proteins, there are a number of intrinsically disordered proteins (IDPs) that can also serve as highly effective protecting agents in the cells. One of the largest groups of disordered chaperones is the group of dehydrins, proteins that are expressed at high levels under different abiotic stress conditions, such as drought, high temperature, or osmotic stress. Dehydrins are characterized by the presence of different conserved sequence motifs that also serve as the basis for their categorization. Despite their accepted importance, the exact role and relevance of the conserved regions have not yet been formally addressed. Here, we explored the involvement of each conserved segment in the protective function of the intrinsically disordered stress protein (IDSP) A. thaliana's Early Response to Dehydration (ERD14). We show that segments that are directly involved in partner binding, and others that are not, are equally necessary for proper function and that cellular protection emerges from the balanced interplay of different regions of ERD14.

Novel Roles of SH2 and SH3 Domains in Lipid Binding.

Signal transduction, the ability of cells to perceive information from the surroundings and alter behavior in response, is an essential property of life. Studies on tyrosine kinase action fundamentally changed our concept of cellular regulation. The induced assembly of subcellular hubs via the recognition of local protein or lipid modifications by modular protein interactions is now a central paradigm in signaling. Such molecular interactions are mediated by specific protein interaction domains. The first such domain identified was the SH2 domain, which was postulated to be a reader capable of finding and binding protein partners displaying phosphorylated tyrosine side chains. The SH3 domain was found to be involved in the formation of stable protein sub-complexes by constitutively attaching to proline-rich surfaces on its binding partners. The SH2 and SH3 domains have thus served as the prototypes for a diverse collection of interaction domains that recognize not only proteins but also lipids, nucleic acids, and small molecules. It has also been found that particular SH2 and SH3 domains themselves might also bind to and rely on lipids to modulate complex assembly. Some lipid-binding properties of SH2 and SH3 domains are reviewed here.

Recent Updates on the Significance of KRAS Mutations in Colorectal Cancer Biology.

The most commonly mutated isoform of RAS among all cancer subtypes is KRAS. In this review, we focus on the special role of KRAS mutations in colorectal cancer (CRC), aiming to collect recent data on KRAS-driven enhanced cell signalling, in vitro and in vivo research models, and CRC development-related processes such as metastasis and cancer stem cell formation. We attempt to cover the diverse nature of the effects of KRAS mutations on age-related CRC development. As the incidence of CRC is rising in young adults, we have reviewed the driving forces of ageing-dependent CRC.

Solution NMR Structure of the SH3 Domain of Human Caskin1 Validates the Lack of a Typical Peptide Binding Groove and Supports a Role in Lipid Mediator Binding.

SH3 domains constitute an important class of protein modules involved in a variety of cellular functions. They participate in protein-protein interactions via their canonical ligand binding interfaces composed of several evolutionarily conserved aromatic residues forming binding grooves for typical (PxxP) and atypical (PxxxPR, RxxK, RKxxY) binding motifs. The calcium/calmodulin-dependent serine protein kinase (CASK)-interacting protein 1, or Caskin1, a multidomain scaffold protein regulating the cortical actin filaments, is enriched in neural synapses in mammals. Based on its known interaction partners and knock-out animal studies, Caskin1 may play various roles in neural function and it is thought to participate in several pathological processes of the brain. Caskin1 has a single, atypical SH3 domain in which key aromatic residues are missing from the canonical binding groove. No protein interacting partner for this SH3 domain has been identified yet. Nevertheless, we have recently demonstrated the specific binding of this SH3 domain to the signaling lipid mediator lysophospatidic acid (LPA) in vitro. Here we report the solution NMR structure of the human Caskin1 SH3 domain and analyze its structural features in comparison with other SH3 domains exemplifying different strategies in target selectivity. The key differences revealed by our structural study show that the canonical binding groove found in typical SH3 domains accommodating proline-rich motifs is missing in Caskin1 SH3, most likely excluding a bona fide protein target for the domain. The LPA binding site is distinct from the altered protein binding groove. We conclude that the SH3 domain of Caskin1 might mediate the association of Caskin1 with membrane surfaces with locally elevated LPA content.

Advances in Understanding TKS4 and TKS5: Molecular Scaffolds Regulating Cellular Processes from Podosome and Invadopodium Formation to Differentiation and Tissue Homeostasis.

Scaffold proteins are typically thought of as multi-domain "bridging molecules." They serve as crucial regulators of key signaling events by simultaneously binding multiple participants involved in specific signaling pathways. In the case of epidermal growth factor (EGF)-epidermal growth factor receptor (EGFR) binding, the activated EGFR contacts cytosolic SRC tyrosine-kinase, which then becomes activated. This process leads to the phosphorylation of SRC-substrates, including the tyrosine kinase substrates (TKS) scaffold proteins. The TKS proteins serve as a platform for the recruitment of key players in EGFR signal transduction, promoting cell spreading and migration. The TKS4 and the TKS5 scaffold proteins are tyrosine kinase substrates with four or five SH3 domains, respectively. Their structural features allow them to recruit and bind a variety of signaling proteins and to anchor them to the cytoplasmic surface of the cell membrane. Until recently, TKS4 and TKS5 had been recognized for their involvement in cellular motility, reactive oxygen species-dependent processes, and embryonic development, among others. However, a number of novel functions have been discovered for these molecules in recent years. In this review, we attempt to cover the diverse nature of the TKS molecules by discussing their structure, regulation by SRC kinase, relevant signaling pathways, and interaction partners, as well as their involvement in cellular processes, including migration, invasion, differentiation, and adipose tissue and bone homeostasis. We also describe related pathologies and the established mouse models.

Novel regulation of Ras proteins by direct tyrosine phosphorylation and dephosphorylation.

Somatic mutations in the RAS genes are frequent in human tumors, especially in pancreatic, colorectal, and non-small-cell lung cancers. Such mutations generally decrease the ability of Ras to hydrolyze GTP, maintaining the protein in a constitutively active GTP-bound form that drives uncontrolled cell proliferation. Efforts to develop drugs that target Ras oncoproteins have been unsuccessful. Recent emerging data suggest that Ras regulation is more complex than the scientific community has believed for decades. In this review, we summarize advances in the "textbook" view of Ras activation. We also discuss a novel type of Ras regulation that involves direct phosphorylation and dephosphorylation of Ras tyrosine residues. The discovery that pharmacological inhibition of the tyrosine phosphoprotein phosphatase SHP2 maintains mutant Ras in an inactive state suggests that SHP2 could be a novel drug target for the treatment of Ras-driven human cancers.

Interplay of Structural Disorder and Short Binding Elements in the Cellular Chaperone Function of Plant Dehydrin ERD14.

Details of the functional mechanisms of intrinsically disordered proteins (IDPs) in living cells is an area not frequently investigated. Here, we dissect the molecular mechanism of action of an IDP in cells by detailed structural analyses based on an in-cell nuclear magnetic resonance experiment. We show that the ID stress protein (IDSP) A. thaliana Early Response to Dehydration (ERD14) is capable of protecting E. coli cells under heat stress. The overexpression of ERD14 increases the viability of E. coli cells from 38.9% to 73.9% following heat stress (50 °C × 15 min). We also provide evidence that the protection is mainly achieved by protecting the proteome of the cells. In-cell NMR experiments performed in E. coli cells show that the protective activity is associated with a largely disordered structural state with conserved, short sequence motifs (K- and H-segments), which transiently sample helical conformations in vitro and engage in partner binding in vivo. Other regions of the protein, such as its S segment and its regions linking and flanking the binding motifs, remain unbound and disordered in the cell. Our data suggest that the cellular function of ERD14 is compatible with its residual structural disorder in vivo.

The effects of mutant Ras proteins on the cell signalome.

The genetic alterations in cancer cells are tightly linked to signaling pathway dysregulation. Ras is a key molecule that controls several tumorigenesis-related processes, and mutations in RAS genes often lead to unbiased intensification of signaling networks that fuel cancer progression. In this article, we review recent studies that describe mutant Ras-regulated signaling routes and their cross-talk. In addition to the two main Ras-driven signaling pathways, i.e., the RAF/MEK/ERK and PI3K/AKT/mTOR pathways, we have also collected emerging data showing the importance of Ras in other signaling pathways, including the RAC/PAK, RalGDS/Ral, and PKC/PLC signaling pathways. Moreover, microRNA-regulated Ras-associated signaling pathways are also discussed to highlight the importance of Ras regulation in cancer. Finally, emerging data show that the signal alterations in specific cell types, such as cancer stem cells, could promote cancer development. Therefore, we also cover the up-to-date findings related to Ras-regulated signal transduction in cancer stem cells.

K-Ras prenylation as a potential anticancer target.

KRAS is one of the most commonly mutated oncogene and a negative predictive factor for a number of targeted therapies. Therefore, the development of targeting strategies against mutant KRAS is urgently needed. One potential strategy involves disruption of K-Ras membrane localization, which is necessary for its proper function. In this review, we summarize the current data about the importance of membrane-anchorage of K-Ras and provide a critical evaluation of this targeting paradigm focusing mainly on prenylation inhibition. Additionally, we performed a RAS mutation-specific analysis of prenylation-related drug sensitivity data from a publicly available database ( https://depmap.org/repurposing/ ) of three classes of prenylation inhibitors: statins, N-bisphosphonates, and farnesyl-transferase inhibitors. We observed significant differences in sensitivity to N-bisphosphonates and farnesyl-transferase inhibitors depending on KRAS mutational status and tissue of origin. These observations emphasize the importance of factors affecting efficacy of prenylation inhibition, like distinct features of different KRAS mutations, tissue-specific mutational patterns, K-Ras turnover, and changes in regulation of prenylation process. Finally, we enlist the factors that might be responsible for the large discrepancy between the outcomes in preclinical and clinical studies including methodological pitfalls, the incomplete understanding of K-Ras protein turnover, and the variation of KRAS dependency in KRAS mutant tumors.

Discovery of a novel kinase hinge binder fragment by dynamic undocking.

One of the key motifs of type I kinase inhibitors is their interactions with the hinge region of ATP binding sites. These interactions contribute significantly to the potency of the inhibitors; however, only a tiny fraction of the available chemical space has been explored with kinase inhibitors reported in the last twenty years. This paper describes a workflow utilizing docking with rDock and dynamic undocking (DUck) for the virtual screening of fragment libraries in order to identify fragments that bind to the kinase hinge region. We have identified 8-amino-2H-isoquinolin-1-one (MR1), a novel and potent hinge binding fragment, which was experimentally tested on a diverse set of kinases, and is hereby suggested for future fragment growing or merging efforts against various kinases, particularly MELK. Direct binding of MR1 to MELK was confirmed by STD-NMR, and its binding to the ATP-pocket was confirmed by a new competitive binding assay based on microscale thermophoresis.

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.

Analysis of Tks4 Knockout Mice Suggests a Role for Tks4 in Adipose Tissue Homeostasis in the Context of Beigeing.

Obesity and adipocyte malfunction are related to and arise as consequences of disturbances in signaling pathways. Tyrosine kinase substrate with four Src homology 3 domains (Tks4) is a scaffold protein that establishes a platform for signaling cascade molecules during podosome formation and epidermal growth factor receptor (EGFR) signaling. Several lines of evidence have also suggested that Tks4 has a role in adipocyte biology; however, its roles in the various types of adipocytes at the cellular level and in transcriptional regulation have not been studied. Therefore, we hypothesized that Tks4 functions as an organizing molecule in signaling networks that regulate adipocyte homeostasis. Our aims were to study the white and brown adipose depots of Tks4 knockout (KO) mice using immunohistology and western blotting and to analyze gene expression changes regulated by the white, brown, and beige adipocyte-related transcription factors via a PCR array. Based on morphological differences in the Tks4-KO adipocytes and increased uncoupling protein 1 (UCP1) expression in the white adipose tissue (WAT) of Tks4-KO mice, we concluded that the beigeing process was more robust in the WAT of Tks4-KO mice compared to the wild-type animals. Furthermore, in the Tks4-KO WAT, the expression profile of peroxisome proliferator-activated receptor gamma (PPARγ)-regulated adipogenesis-related genes was shifted in favor of the appearance of beige-like cells. These results suggest that Tks4 and its downstream signaling partners are novel regulators of adipocyte functions and PPARγ-directed white to beige adipose tissue conversion.

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.