Richard A. Klinghoffer

Src family kinases are required for integrin but not PDGFR signal transduction

Src family kinases (SFKs) have been implicated as important regulators of ligand‐induced cellular responses including proliferation, survival, adhesion and migration. Analysis of SFK function has been impeded by extensive redundancy between family members. We have generated mouse embryos harboring functional null mutations of the ubiquitously expressed SFKs Src, Yes and Fyn. This triple mutation leads to severe developmental defects and lethality by E9.5. To elucidate the molecular mechanisms underlying this phenotype, SYF cells (deficient for Src, Yes and Fyn) were derived and tested for their ability to respond to growth factors or plating on extracellular matrix. Our studies reveal that while Src, Yes and Fyn are largely dispensable for platelet‐derived growth factor (PDGF)‐induced signaling, they are absolutely required to mediate specific functions regulated by extracellular matrix proteins. Fibronectin‐induced tyrosine phosphorylation of focal adhesion proteins, including the focal adhesion kinase FAK, was nearly eliminated in the absence of Src, Yes and Fyn. Furthermore, consistent with previous reports demonstrating the importance of FAK for cell migration, SYF cells displayed reduced motility in vitro. These results demonstrate that SFK activity is essential during embryogenesis and suggest that defects observed in SYF triple mutant embryos may be linked to deficiencies in signaling by extracellular matrix‐coupled receptors.

Evolutionary divergence of platelet-derived growth factor alpha receptor signaling mechanisms.

ABSTRACT Receptor tyrosine kinases (RTKs) direct diverse cellular and developmental responses by stimulating a relatively small number of overlapping signaling pathways. Specificity may be determined by RTK expression patterns or by differential activation of individual signaling pathways. To address this issue we generated knock-in mice in which the extracellular domain of the mouse platelet-derived growth factor alpha receptor (PDGFαR) is fused to the cytosolic domain of Drosophila Torso (αTor) or the mouse fibroblast growth factor receptor 1 (αFR). αTor homozygous embryos exhibit significant rescue of neural crest and angiogenesis defects normally found in PDGFαR-null embryos yet fail to rescue skeletal or extraembryonic defects. This phenotype was associated with the ability of αTor to stimulate the mitogen-activated protein (MAP) kinase pathway to near wild-type levels but failure to completely activate other pathways, such as phosphatidylinositol (PI) 3-kinase. The αFR chimeric receptor fails to rescue any aspect of the PDGFαR-null phenotype. Instead, αFR expression leads to a gain-of-function phenotype highlighted by ectopic bone development. The αFR phenotype was associated with a failure to limit MAP kinase signaling and to engage significant PI3-kinase response. These results suggest that precise regulation of divergent downstream signaling pathways is critical for specification of RTK function.

An Allelic Series at the PDGFαR Locus Indicates Unequal Contributions of Distinct Signaling Pathways During Development

A central issue in signal transduction is the physiological contribution of different growth factor-initiated signaling pathways. We have generated knockin mice harboring mutations in the PDGFalpha receptor (PDGFalphaR) that selectively eliminate its capacity to activate PI3 kinase (alpha(PI3K)) or Src family kinases (alpha(Src)). The alpha(PI3K) mutation leads to neonatal lethality due to impaired signaling in many cell types, but the alpha(Src) mutation only affects oligodendrocyte development. A third knockin line containing mutations that eliminate multiple docking sites does not increase the severity of the alpha(PI3K) mutation. However, embryos with mutations in the PI3K binding sites of both PDGFRs (alpha and beta) recapitulate the PDGFalphaR null phenotype. Our results indicate that PI3K has a predominant role in PDGFalphaR signaling in vivo and that RTK-activated signaling pathways execute both specific and overlapping functions during mammalian development.

The Two PDGF Receptors Maintain Conserved Signaling In Vivo despite Divergent Embryological Functions

Gene targeting studies have indicated that the two receptors for PDGF, alpha and beta, direct unique functions during development. Distinct ligand affinities, patterns of gene expression, and/or mechanisms of signal relay may account for functional specificity of the two PDGF receptor isoforms. To distinguish between these factors, we have created two complementary lines of knockin mice in which the intracellular signaling domains of one PDGFR have been removed and replaced by those of the other PDGFR. While both lines demonstrated substantial rescue of normal development, substitution of the PDGFbetaR signaling domains with those of the PDGFalphaR resulted in varying degrees of vascular disease. This observation provides a framework for discussing the evolution of receptor tyrosine kinase functional specificity.

Src Catalytic but Not Scaffolding Function Is Needed for Integrin-Regulated Tyrosine Phosphorylation, Cell Migration, and Cell Spreading

ABSTRACT Src family kinases (SFKs) are crucial for signaling through a variety of cell surface receptors, including integrins. There is evidence that integrin activation induces focal adhesion kinase (FAK) autophosphorylation at Y397 and that Src binds to and is activated by FAK to carry out subsequent phosphorylation events. However, it has also been suggested that Src functions as a scaffolding molecule through its SH2 and SH3 domains and that its kinase activity is not necessary. To examine the role of SFKs in integrin signaling, we have expressed various Src molecules in fibroblasts lacking other SFKs. In cells plated on fibronectin, FAK could indeed autophosphorylate at Y397 independently of Src but with lower efficiency than when Src was present. This step was promoted by kinase-inactive Src, but Src kinase activity was required for full rescue. Src kinase activity was also required for phosphorylation of additional sites on FAK and for other integrin-directed functions, including cell migration and spreading on fibronectin. In contrast, Src mutations in the SH2 or SH3 domain greatly reduced binding to FAK, Cas, and paxillin but had little effect on tyrosine phosphorylation or biological assays. Furthermore, our indirect evidence indicates that Src kinase activity does not need to be regulated to promote cell migration and FAK phosphorylation. Although Src clearly plays important roles in integrin signaling, it was not concentrated in focal adhesions. These results indicate that the primary role of Src in integrin signaling is as a kinase. Indirect models for Src function are proposed.

PDGF induces an early and a late wave of PI 3-kinase activity, and only the late wave is required for progression through G1

BACKGROUND Platelet-derived growth factor (PDGF) triggers cytoskeletal rearrangements and chemotaxis within minutes. These events are at least in part due to the activation of phosphoinositide (PI) 3-kinase; there is good temporal correlation between these events and the accumulation of 3-phosphorylated products of the kinase. Prolonged and continuous PDGF exposure results in S-phase entry many hours after the initial burst of activity. Although early signals appear responsible for the early responses, they may not fully account for later responses, such as cell-cycle progression. RESULTS We assessed when PI 3-kinase products accumulate in PDGF-stimulated cells. In addition to the previously identified early accumulation of products, we detected a second, prolonged wave of accumulation 3-7 hours after stimulation. To determine the relative contribution of each phase to PDGF-dependent DNA synthesis, we first developed an assay in which synthetic 3-phosphorylated lipids were used to rescue DNA synthesis in cells expressing a PDGF-receptor mutant. The lipids rescued DNA synthesis only when added 2-6 hours after PDGF. In addition, PI 3-kinase inhibitors failed to block PDGF-dependent DNA synthesis if added during the first wave of PI 3-kinase activity, but adding them later, in G1 phase, prevented PDGF-dependent cell-cycle progression. CONCLUSIONS PDGF induces distinct waves of PI 3-kinase activity. The second wave is required for PDGF-dependent DNA synthesis, whereas the initial wave is not. One of the ways in which cells use PI 3-kinase to mediate distinct cellular responses seems to be by regulating when its products accumulate.

Platelet-derived growth factor-dependent activation of phosphatidylinositol 3-kinase is regulated by receptor binding of SH2-domain-containing proteins which influence Ras activity.

Upon binding of platelet-derived growth factor (PDGF), the PDGF beta receptor (PDGFR) undergoes autophosphorylation on distinct tyrosine residues and binds several SH2-domain-containing signal relay enzymes, including phosphatidylinositol 3-kinase (PI3K), phospholipase C gamma (PLC gamma), the GTPase-activating protein of Ras (RasGAP), and the tyrosine phosphatase SHP-2. In this study, we have investigated whether PDGF-dependent PI3K activation is affected by the other proteins that associate with the PDGFR. We constructed and characterized a series of PDGFR mutants which contain binding sites for PI3K as well as one additional protein, either RasGAP, SHP-2, or PLC gamma. While all of the receptors had wild-type levels of PDGF-stimulated tyrosine kinase activity and associated with comparable amounts of PI3K activity, their abilities to trigger accumulation of PI3K products in vivo differed dramatically. The wild-type receptor, as well as receptors that recruited PI3K or PI3K and SHP-2, were all capable of fully activating PI3K. In contrast, receptors that associated with PI3K and RasGAP or PI3K and PLC gamma displayed a greatly reduced ability to stimulate production of PI3K products. When this series of receptors was tested for their ability to activate Ras, we observed a strong positive correlation between Ras activation and PI3K activation. Further investigation of the relationship between Ras and PI3K indicated that Ras was upstream of PI3K. Thus, activation of PI3K requires not only binding of PI3K to the tyrosine-phosphorylated PDGFR but accumulation of GTP-bound Ras as well. Furthermore, PLC gamma and RasGAP negatively modulate PDGF-dependent PI3K activation. Finally, PDGF-stimulated signal relay can be regulated by altering the ratio of SH2-domain-containing enzymes that are recruited to the PDGFR.

Growth factor signaling pathways in vascular development

Recent research on the formation and maintenance of the vasculature in the embryo and in the adult has provided a greater understanding of the cellular signals involved in these processes. With this understanding comes the potential means of controlling vascularization in pathological situations such as tumorigenesis and wounding. For the purpose of this review, we will discuss the key receptor tyrosine kinases involved in vascular function and the molecules which relay signals downstream of receptor activation. The receptor tyrosine kinases discussed include the vascular endothelial cell growth factor receptors, Eph receptors, Tie1, and Tie2, all of which are expressed on vascular endothelial cells. We also discuss the roles of the platelet derived growth factor receptors which are expressed on vascular smooth muscle cells. While all of these receptor tyrosine kinases activate many similar effector molecules, some of the signals initiated appear to be distinct. This may explain, at least in part, how different receptor tyrosine kinases expressed in overlapping patterns on the developing vasculature, direct unique biological functions.

A modified gene trap approach for improved high-throughput cancer drug discovery

While advances in laboratory automation has dramatically increased throughout of compound screening efforts, development of robust cell-based assays in relevant disease models remain resource-intensive and time-consuming, presenting a bottleneck to drug discovery campaigns. To address this issue, we present a modified gene trap approach to efficiently generate pathway-specific reporters that result in a robust “on” signal when the pathway of interest is inhibited. In this proof-of-concept study, we used vemurafenib and trametinib to identify traps that specifically detect inhibition of the mitogen-activated protein kinase (MAPK) pathway in a model of BRAFV600E driven human malignant melanoma. We demonstrate that insertion of our trap into particular loci results in remarkably specific detection of MAPK pathway inhibitors over compounds targeting any other pathway or cellular function. The accuracy of our approach was highlighted in a pilot screen of ~6000 compounds where 40 actives were detected, including 18 MEK, 10 RAF, and 3 ERK inhibitors along with a few compounds representing previously under-characterized inhibitors of the MAPK pathway. One such compound, bafetinib, a second generation BCR/ABL inhibitor, reduced phosphorylation of ERK and when combined with trametinib, both in vitro and in vivo, reduced growth of vemurafenib resistant melanoma cells. While piloted in a model of BRAF-driven melanoma, our results set the stage for using this approach to rapidly generate reporters against any transcriptionally active pathway across a wide variety of disease-relevant cell-based models to expedite drug discovery efforts.