Activation Of Src Family Kinases Research Articles

YES, a Src Family Kinase, Is a Proximal Glucose-specific Activator of Cell Division Cycle Control Protein 42 (Cdc42) in Pancreatic Islet β Cells

Second-phase insulin secretion sustains insulin release in the face of hyperglycemia associated with insulin resistance, requiring the continued mobilization of insulin secretory granules to the plasma membrane. Cdc42, the small Rho family GTPase recognized as the proximal glucose-specific trigger to elicit second-phase insulin secretion, signals downstream to activate the p21-activated kinase (PAK1), which then signals to Raf-1/MEK/ERK to induce filamentous actin (F-actin) remodeling, to ultimately mobilize insulin granules to the plasma membrane. However, the steps required to initiate Cdc42 activation in a glucose-specific manner in β cells have remained elusive. Toward this, we identified the involvement of the Src family kinases (SFKs), based upon the ability of SFK inhibitors to block glucose-stimulated Cdc42 and PAK1 activation events as well as the amplifying pathway of glucose-stimulated insulin release, in MIN6 β cells. Indeed, subsequent studies performed in human islets revealed that SFK phosphorylation was induced only by glucose and within 1 min of stimulation before the activation of Cdc42 at 3 min. Furthermore, pervanadate treatment validated the phosphorylation event to be tyrosine-specific. Although RT-PCR showed β cells to express five different SFK proteins, only two of these, YES and Fyn kinases, were found localized to the plasma membrane, and of these two, only YES kinase underwent glucose-stimulated tyrosine phosphorylation. Immunodetection and RNAi analyses further established YES kinase as a proximal glucose-specific signal in the Cdc42-signaling cascade. Identification of YES kinase provides new insight into the mechanisms underlying the sustainment of insulin secretion via granule mobilization/replenishment and F-actin remodeling.

Potentiation of TNF‐induced inflammatory transcriptional regulation by SFK activation (278.3)

TNF induces dramatic changes in the transcriptional profile of endothelial cells to promote a pro‐inflammatory phenotype that allows leukocyte transendothelial migration and loss of barrier function. We have previously shown that treatment with 50 pg/ml TNF increases endothelial permeability after 4‐6 h in monolayers of human dermal microvascular endothelial cells expressing DN‐Csk to activate endogenous Src Family Kinases (SFKs) but not in control (LacZ) monolayers. We thus reasoned that this delay in increasing permeability could be due to a requirement in transcriptional changes. Here, we demonstrate that activation of SFKs potentiates the transcriptional effects induced by TNF. After 6 h, TNF elevated ICAM1, E‐Selectin, HSPA1A/B and complement C3 mRNA levels 41‐, 76‐, 12‐ and 24‐fold respectively. In the presence of DN‐Csk, the same treatment induced these genes 149‐, 310‐, 38‐ and 202‐fold, respectively. Similar effects were observed in the mRNA of TNF itself (46 vs 193 fold) and lymphotoxin A (5 vs 21 fold), but not lymphotoxin B (86 vs 83 fold). ATF3 was induced 7 and 20‐fold respectively. EGR2 mRNA was induced 5‐fold by DN‐Csk and 9‐fold by the dual treatment, while Fos mRNA was induced 4‐fold in DN‐Csk‐expressing cells after 6h TNF and 9‐fold after 18 h. While these treatments did not lead to changes in the expression of many junctional genes, ZO‐1 and VE‐cadherin localization was drastically altered as determined by immunofluorescence. A pro‐inflammatory transcriptional profile, together with local posttranslational changes at the cell‐cell junctions, could explain the synergistic loss of barrier function observed with dual TNF signaling and endogenous SFK activation.Grant Funding Source: Supported by an AHA SDG grant 13SDG17100110 to APA.

PTK7-Src Signaling at Epithelial Cell Contacts Mediates Spatial Organization of Actomyosin and Planar Cell Polarity

Actomyosin contractility plays a key role in tissue morphogenesis. During mammalian development, PTK7 regulates epithelial morphogenesis and planar cell polarity (PCP) through modulation of actomyosin contractility, but the underlying mechanism is unknown. Here, we show that PTK7 interacts with the tyrosine kinase Src and stimulates Src signaling along cell-cell contacts. We further identify ROCK2 as a target of junctional PTK7-Src signaling. PTK7 knockdown in cultured epithelial cells reduced the level of active Src at cell-cell contacts, resulting in delocalization of ROCK2 from cell-cell contacts and decreased junctional contractility, with a concomitant increase in actomyosin on the basal surface. Moreover, we present in vivo evidence that Src family kinase (SFK) activity is critical for PCP regulation in the auditory sensory epithelium and that PTK7-SFK signaling regulates tyrosine phosphorylation of junctional ROCK2. Together, these results delineate a PTK7-Src signaling module for spatial regulation of ROCK activity, actomyosin contractility, and epithelial PCP.

Synthesis and structure–activity analysis of diphenylpyrazolodiazene inhibitors of the HIV-1 Nef virulence factor

HIV-1 Nef is a critical AIDS progression factor yet underexplored target for antiretroviral drug discovery. A recent high-throughput screen for pharmacological inhibitors of Nef-dependent Src-family kinase activation identified a diphenylpyrazolodiazene hit compound with submicromolar potency in HIV-1 replication assays against a broad range of primary Nef variants. This compound, known as ‘B9’, binds directly to Nef and inhibits its dimerization in cells as a possible mechanism of action. Here were synthesized a diverse set of B9 analogs and identified structural features essential to antiretroviral activity. Chemical modifications to each of the three rings present in the parent compound were identified that did not compromise antiviral action. These analogs will guide the development of next-generation compounds with appropriate pharmacological profiles for assessment of antiretroviral activity in vivo.

Src tyrosine kinase signaling antagonizes nuclear localization of FOXO and inhibits its transcription factor activity

Biochemical experiments in mammalian cells have linked Src family kinase activity to the insulin signaling pathway. To explore the physiological link between Src and a central insulin pathway effector, we investigated the effect of different Src signaling levels on the Drosophila transcription factor dFOXO in vivo. Ectopic activation of Src42A in the starved larval fatbody was sufficient to drive dFOXO out of the nucleus. When Src signaling levels were lowered by means of loss-of-function mutations or pharmacological inhibition, dFOXO localization was shifted to the nucleus in growing animals, and transcription of the dFOXO target genes d4E-BP and dInR was induced. dFOXO loss-of-function mutations rescued the induction of dFOXO target gene expression and the body size reduction of Src42A mutant larvae, establishing dFOXO as a critical downstream effector of Src signaling. Furthermore, we provide evidence that the regulation of FOXO transcription factors by Src is evolutionarily conserved in mammalian cells.

PKA and CaMKII mediate PI3K activation in bovine sperm by inhibition of the PKC/PP1 cascade

To enable fertilization, spermatozoa must undergo several biochemical processes in the female reproductive tract, collectively called capacitation. These processes involve protein kinase A (PKA)-dependent protein tyrosine phosphorylation including phosphatidylinositol-3-kinase (PI3K). It is not known how PKA, a serine/threonine (S/T) kinase, mediates tyrosine phosphorylation of proteins. We recently showed that inhibition of S/T phosphatase 1 (PP1) causes a significant increase in phospho-PI3K. In this study, we propose a mechanism by which PKA and PP1 mediate an increase in PI3K tyrosine phosphorylation and implicate calmodulin-dependent kinase II (CaMKII) in this process. Inhibition of sperm PP1 or PKC, stimulated CaMKII phosphorylation/activation, and inhibition of PKC enhanced PP1 phosphorylation/inactivation. Inhibition of CaMKII, using KN-93, caused significant reduction in phospho-PP1, indicating its activation. Moreover, KN-93 prevented the dephosphorylation/inactivation of PKC. We therefore suggest that CaMKII inhibits PKC, leading to PP1 inhibition and the reciprocal auto-activation of CaMKII. Thus, CaMKII can regulate its own activation by inhibiting the PKC/PP1 cascade. Inhibition of Src family kinases (SFK) caused significant inhibition of CaMKII and PP1 phosphorylation, suggesting that SFK activity results in PP1 inhibition and CaMKII activation. Activation of sperm PKA by 8Br-cAMP revealed an increase in phospho-CaMKII, which was inhibited by PKA inhibitor. Tyrosine phosphorylation of PI3K was stimulated by 8Br-cAMP and by PKC or PP1 inhibition and was abrogated by CaMKII inhibition. Furthermore, phosphorylation/activation of the tyrosine kinase Pyk2 was enhanced by PP1 inhibition, and this activation is blocked by CaMKII inhibition. Thus, PKA activates Src, which inhibits PP1, leading to CaMKII and Pyk2 activation, resulting in PI3K tyrosine phosphorylation/activation.

Long-term treatment with PP2 after spinal cord injury resulted in functional locomotor recovery and increased spared tissue.

The spinal cord has the ability to regenerate but the microenvironment generated after trauma reduces that capacity. An increase in Src family kinase (SFK) activity has been implicated in neuropathological conditions associated with central nervous system trauma. Therefore, we hypothesized that a decrease in SFK activation by a long-term treatment with 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyramidine (PP2), a selective SFK inhibitor, after spinal cord contusion with the New York University (NYU) impactor device would generate a permissive environment that improves axonal sprouting and/or behavioral activity. Results demonstrated that long-term blockade of SFK activation with PP2 increases locomotor activity at 7, 14, 21 and 28 days post-injury in the Basso, Beattie, and Bresnahan open field test, round and square beam crossing tests. In addition, an increase in white matter spared tissue and serotonin fiber density was observed in animals treated with PP2. However, blockade of SFK activity did not change the astrocytic response or infiltration of cells from the immune system at 28 days post-injury. Moreover, a reduced SFK activity with PP2 diminished Ephexin (a guanine nucleotide exchange factor) phosphorylation in the acute phase (4 days post-injury) after trauma. Together, these findings suggest a potential role of SFK in the regulation of spared tissue and/or axonal outgrowth that may result in functional locomotor recovery during the pathophysiology generated after spinal cord injury. Our study also points out that ephexin1 phosphorylation (activation) by SFK action may be involved in the repulsive microenvironment generated after spinal cord injury.

PSTPIP2 dysregulation contributes to aberrant terminal differentiation in GATA-1-deficient megakaryocytes by activating LYN

GATA1 mutations are tightly associated with transient myeloproliferative disorder (TMD) and acute megakaryoblstic leukemia (AMKL) in children with Down syndrome. Numerous genes are altered in GATA-1-deficient megakaryocytes, which may contribute to the hyperproliferation and abnormal terminal differentiation of these malignant cells. In this study, we demonstrate that Pstpip2 is a GATA-1-repressed gene that controls megakaryopoiesis. Ectopic expression of PSTPIP2 impaired megakaryocytic differentiation as evidenced by a decrease of CD41 expression and reduced DNA content in K562 cells. PSTPIP2 overexpression also caused enhanced activation of Src family kinases and subsequently reduced ERK phosphorylation. Consistently, PSTPIP2 knockdown showed the opposite effect on differentiation and signaling. Moreover, the W232A mutant of PSTPIP2, defective in its interaction with PEST family phosphatases that recruit c-Src terminal kinase (CSK) to suppress Src family kinases, failed to inhibit differentiation and lost its ability to enhance Src family kinases or reduce ERK phosphorylation. In fact, the W232A mutant of PSTPIP2 promoted megakaryocyte differentiation. These observations suggest that PSTPIP2 recruiting PEST phosphatases somehow blocked CSK activity and led to enhanced activation of Src family kinases and reduced ERK phosphorylation, which ultimately repressed megakaryocyte differentiation. Supporting this idea, PSTPIP2 interacted with LYN and the expression of a dominant negative LYN (LYN DN) overwhelmed the inhibitory effect of PSTPIP2 on differentiation and ERK signaling. In addition, a constitutively active LYN (LYN CA) normalized the enhanced megakaryocyte differentiation and repressed ERK signaling in PSTPIP2 knockdown cells. Finally, we found that PSTPIP2 repressed ERK signaling, differentiation, and proliferation and verified that PSTPIP2 upregulation repressed megakaryocyte development in primary mouse bone marrow cells. Our study thus reveals a novel mechanism by which dysregulation of PSTPIP2 due to GATA-1 deficiency may contribute to abnormal megakaryocyte proliferation and differentiation in pathogenesis of related diseases.

A Functional Interplay between the Small GTPase Rab11a and Mitochondria-shaping Proteins Regulates Mitochondrial Positioning and Polarization of the Actin Cytoskeleton Downstream of Src Family Kinases

It is believed that mitochondrial dynamics is coordinated with endosomal traffic rates during cytoskeletal remodeling, but the mechanisms involved are largely unknown. The adenovirus early region 4 ORF4 protein (E4orf4) subverts signaling by Src family kinases (SFK) to perturb cellular morphology, membrane traffic, and organellar dynamics and to trigger cell death. Using E4orf4 as a model, we uncovered a functional connection between mitochondria-shaping proteins and the small GTPase Rab11a, a key regulator of polarized transport via recycling endosomes. We found that E4orf4 induced dramatic changes in the morphology of mitochondria along with their mobilization at the vicinity of a polarized actin network typifying E4orf4 action, in a manner controlled by SFK and Rab11a. Mitochondrial remodeling was associated with increased proximity between Rab11a and mitochondrial membranes, changes in fusion-fission dynamics, and mitochondrial relocalization of the fission factor dynamin-related protein 1 (Drp1), which was regulated by the Rab11a effector protein FIP1/RCP. Knockdown of FIP1/RCP or inhibition of Drp1 markedly impaired mitochondrial remodeling and actin assembly, involving Rab11a-mediated mitochondrial dynamics in E4orf4-induced signaling. A similar mobilization of mitochondria near actin-rich structures was mediated by Rab11 and Drp1 in viral Src-transformed cells and contributed to the biogenesis of podosome rosettes. These findings suggest a role for Rab11a in the trafficking of Drp1 to mitochondria upon SFK activation and unravel a novel functional interplay between Rab11a and mitochondria during reshaping of the cell cytoskeleton, which would facilitate mitochondria redistribution near energy-requiring actin-rich structures.

N-WASP-directed actin polymerization activates p130Cas phosphorylation and lamellipodium spreading

Tyrosine phosphorylation of the substrate domain of Cas (CasSD) correlates with increased cell migration in healthy and diseased cells. Here, we address the mechanism leading to the phosphorylation of CasSD in the context of fibronectin-induced early spreading of fibroblasts. We have previously demonstrated that mechanical stretching of CasSD exposes phosphorylation sites for Src family kinases (SFKs). Surprisingly, phosphorylation of CasSD was independent of myosin contractile activity but dependent on actin polymerization. Furthermore, we found that CasSD phosphorylation in the early stages of cell spreading required: (1) integrin anchorage and integrin-mediated activation of SFKs, (2) association of Cas with focal adhesion kinase (FAK), and (3) N-WASP-driven actin-assembly activity. These findings, and analyses of the interactions of the Cas domains, indicate that the N-terminus of Cas associates with the FAK-N-WASP complex at the protrusive edge of the cell and that the C-terminus of Cas associates with the immobilized integrin-SFK cluster. Thus, extension of the leading edge mediated by actin polymerization could stretch Cas during early cell spreading, priming it for phosphorylation.

Inhibition of the kinase Csk in thymocytes reveals a requirement for actin remodeling in the initiation of full TCR signaling.

T cell receptor (TCR) signaling is initiated by Src-family kinases (SFKs). To understand how C-terminal Src kinase (Csk), the negative regulator of SFKs, controls the basal state and the initiation of TCR signaling, we generated mice expressing a PP1-analog inhibitor-sensitive Csk variant (CskAS). Inhibition of CskAS in thymocytes, without TCR engagement, induced potent SFK activation and proximal TCR signaling up to phospholipase C-γ1 (PLC-γ1). Surprisingly, increases in inositol phosphates (InsP), intracellular calcium and Erk phosphorylation were impaired. Altering the actin cytoskeleton pharmacologically or providing CD28 costimulation rescued these defects. Thus, Csk plays a critical role in preventing TCR signaling. However, our studies also revealed a requirement for actin remodeling, initiated by costimulation, for full TCR signaling.

Nitric Oxide Regulation of Na, K‐ATPase Activity in Ocular Ciliary Epithelium Involves Src Family Kinase

The nitric oxide (NO) donor sodium nitroprusside (SNP) is known to reduce aqueous humor (AH) secretion in the isolated porcine eye. Previously, SNP was found to inhibit Na,K-ATPase activity in nonpigmented ciliary epithelium (NPE), AH-secreting cells, through a cGMP/protein kinase G (PKG)-mediated pathway. Here we show Src family kinase (SFK) activation in the Na,K-ATPase activity response to SNP. Ouabain-sensitive (86) Rb uptake was reduced by >35% in cultured NPE cells exposed to SNP (100 µM) or exogenously added cGMP (8-Br-cGMP) (100 µM) and the SFK inhibitor PP2 (10 µM) prevented the response. Ouabain-sensitive ATP hydrolysis was reduced by ~40% in samples detected in material obtained from SNP- and 8-Br-cGMP-treated cells following homogenization, pointing to an intrinsic change of Na,K-ATPase activity. Tyrosine-10 phosphorylation of Na,K-ATPase α1 subunit was detected in SNP and L-arginine-treated cells and the response prevented by PP2. SNP elicited an increase in cell cGMP. Cells exposed to 8-Br-cGMP displayed SFK activation (phosphorylation) and inhibition of both ouabain-sensitive (86) Rb uptake and Na,K-ATPase activity that was prevented by PP2. SFK activation, which also occurred in SNP-treated cells, was suppressed by inhibitors of soluble guanylate cyclase (ODQ; 10 µM) and PKG (KT5823; 1 µM). SNP and 8-Br-cGMP also increased phosphorylation of ERK1/2 and p38 MAPK and the response prevented by PP2. However, U0126 did not prevent SNP or 8-Br-cGMP-induced inhibition of Na,K-ATPase activity. Taken together, the results suggest that NO activates guanylate cyclase to cause a rise in cGMP and subsequent PKG-dependent SFK activation. Inhibition of Na,K-ATPase activity depends on SFK activation.

Src family kinases involved in CXCL12-induced loss of acute morphine analgesia

Functional interactions between the chemokine receptor CXCR4 and opioid receptors have been reported in the brain, leading to a decreased morphine analgesic activity. However the cellular mechanisms responsible for this loss of opioid analgesia are largely unknown. Here we examined whether Src family-kinases (SFK)-linked mechanisms induced by CXCR4 contributed to the loss of acute morphine analgesia and could represent a new physiological anti-opioid signaling pathway. In this way, we showed by immunohistochemistry and western blot that CXCL12 rapidly activated SFK phosphorylation in vitro in primary cultured lumbar rat dorsal root ganglia (DRG) but also in vivo in the DRG and the spinal cord. We showed that SFK activation occurred in a sub population of sensory neurons, in spinal microglia but also in spinal nerve terminals expressing mu-(MOR) and delta-opioid (DOR) receptor. In addition we described that CXCR4 is detected in MOR- and DOR-immunoreactive neurons in the DRG and spinal cord. In vivo, we demonstrated that an intrathecal administration of CXCL12 (1μg) significantly attenuated the subcutaneous morphine (4mg/kg) analgesia. Conversely, pretreatment with a potent CXCR4 antagonist (5μg) significantly enhanced morphine analgesia. Similar effects were obtained after an intrathecal injection of a specific SFK inhibitor, PP2 (10μg). Furthermore, PP2 abrogated CXCL12-induced decrease in morphine analgesia by suppressing SFK activation in the spinal cord. In conclusion, our data highlight that CXCL12-induced loss of acute morphine analgesia is linked to Src family kinases activation.

CD99 Is a Therapeutic Target On Disease Stem Cells In Acute Myeloid Leukemia and The Myelodysplastic Syndromes

The myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are initiated and sustained by self-renewing stem cells. We performed transcriptomal profiling of purified MDS hematopoietic stem cells (HSCs) and identified CD99 as a highly expressed cell surface transcript. Flow cytometry of MDS patient bone marrow (BM) samples (n=63) confirmed that CD99 is frequently increased on MDS HSCs (85%). Assessment of 78 paired diagnosis/relapse AML specimens revealed that CD99 is also frequently overexpressed in AML at diagnosis (81%) and relapse (83%). CD99 is a potential leukemic stem cell (LSC) marker, as CD99 high cells exhibit a CD34+CD38-CD90-CD45RA+ LMPP-like immunophenotype, previously shown to be enriched for LSC activity, whereas CD99 low cells exhibit a CD34+CD38-CD45RA- phenotype resembling normal HSCs and MPPs.Providing additional support that CD99 is a LSC marker in AML, CD99 surface expression is significantly higher in the LSC-enriched CD34+CD38- fraction compared to bulk blasts (p=0.003), as well as in relapsed as compared to diagnostic specimens (p=0.007). To determine if CD99 is preferentially expressed on functional LSCs, we purified the top 10% and bottom 10% of CD99 expressing leukemic blasts from the CD34+CD38-CD90-CD45RA+ fraction of a primary AML specimen. Limiting dilution xenotransplantation experiments demonstrated an estimated LSC frequency of 1 in 24,401 in the top 10% of CD99 expressors, whereas transplantation of up to 360,000 of the bottom 10% of CD99 expressors did not lead to any engraftment. Thus, within the LMPP-like LSC-enriched compartment, LSC activity appears to be restricted to high CD99 expressors.To determine the function of CD99 in AML, we stably transduced MOLM13 AML cells with a CD99 shRNA (8.0-fold knockdown) and xenografted them into NSG mice. Animals transplanted with these cells showed improved survival compared to vector controls (58d vs. 34d, p=0.02). Consistent with its described role in leukocyte trafficking, overexpression of CD99 in AML cell lines promoted transendothelial migration in transwell assays. In primary AML specimens, CD99 expression was higher on PB as compared with BM specimens (p=0.03). Together, these findings suggest that CD99 may promote AML aggressiveness by enhancing transendothelial migration and mobilization.Unexpectedly, CD99 transcript expression in the ECOG 1900 AML patient cohort (n=308) positively correlated with survival (p=0.001). We propose that CD99 may improve survival in the context of chemotherapy by promoting mobilization and thus chemosensitivity of AML cells, and AML cells with low levels of CD99 may exhibit increased retention in chemoprotective BM niches. In line with this hypothesis, the poor prognosis of low CD99 expression appeared to be mitigated by intensification of chemotherapy.To determine whether CD99 may be a relevant therapeutic target, we tested the ability of monoclonal antibodies (mAb) against CD99 to induce direct cytotoxicity in vitro. Anti-CD99 mAbs induced significant cell death in 11 AML and two MDS-derived cell lines, as well as in primary AML blasts (n=7) and MDS HSCs/CD34+ cells (n=3). These mAbs induced cytotoxicity in a CDC and ADCC independent manner, with relative sparing of normal cells expressing low levels of CD99 such as primary human HSCs and endothelial cells, suggesting a mechanism of cytotoxicity unique to AML and MDS. Pre-coating of primary human LSCs with an anti-CD99 mAb prior to transplantation into NSG mice led to impaired engraftment at eight weeks (20% vs. 67%, p=0.009) and improved survival (p=0.05). Immunofluorescence imaging demonstrated that mAb ligation promotes clustering of CD99, and this clustering appears to be critical for cytotoxicity. Accordingly, cross-linking of IgG isotype anti-CD99 mAbs with an anti-IgG secondary antibody enhanced cytotoxicity 3.4-fold. Anti-CD99 mAb treatment was also associated with activation of Src-family kinases, and we propose that anti-CD99 mAbs may promote cytotoxicity by inducing oncogenic stress via dysregulated Src-family kinase activation.Our results establish CD99 as a cell surface marker expressed in AML and MDS stem cells, as a mediator of transendothelial migration, and as a promising therapeutic target for direct targeting by mAbs. Further studies are needed to evaluate the potential of targeting CD99 in AML/MDS in vivo and to further characterize the mechanisms of CD99 mediated cell death. Disclosures:No relevant conflicts of interest to declare.

Src Kinase Can Activate RUNX1 Activity Via Phosphorylation Of C-Terminal Tyrosines and Activated RUNX1 Stimulates Granulopoiesis

We demonstrated that G-CSF signaling in lineage-negative marrow myeloid progenitors induces C-terminal SHP2 tyrosine phosphorylation more potently than does M-CSF signaling and that SHP2 knockdown in marrow or in the 32Dcl3 cell line reduces Cebpa gene transcription and impairs granulopoiesis.1,2 We also found that Runx1 directly activates Cebpa transcription via promoter elements and via a +37 kb enhancer and that Runx1 gene deletion reduces Cebpa mRNA and impairs granulopoiesis.3 Runx1 is phosphorylated by Src kinase on five tyrosines and dephosphorylated by SHP2, and RUNX1(5F) enhances megakaryopoiesis and rescues CD8 T cell development, whereas RUNX1(5D) is ineffective.4 Mutation to F prevents while change to D or E mimics phosphorylation. These findings suggested that in myeloid cells there exists a G-CSF to SHP2 to RUNX1 to Cebpa activation pathway directing granulopoiesis. We compared wild-type (WT) murine RUNX1b and a series of variants, Y260/375/378/379/386F (5F), Y260F (1F), Y375/378/379/386F (4F), 5D, 1D, and 4D for activation in 293T cells of a reporter with four RUNX1-binding sites linked to the TK promoter and the luciferase cDNA. Expression was compared by Western blotting. WT stimulated the reporter 5-fold on average, relative to empty CMV vector, 5D activated the reporter 15-fold, while 5F was nearly inactive. 4F was also nearly inactive, while 4D had WT activity, as did the 1F and 1D variants. Both 4F and 5F were expressed at or above WT levels; interestingly, 5D, but not 4D or 1D, was expressed at much higher levels than the other variants. A similar pattern was obtained with the MCSFR-Luc reporter. To further query the activity of the four C-terminal tyrosines, we evaluated the activity of Y375/378F (2F*), Y379/386F (2F), 2E, Y375F, Y378F, Y379F, and Y386F. The single residue mutations retained WT activity, whereas both the 2F* and 2F variants demonstrated significant reduction despite expression at or above WT levels. The 2E variant expressed at levels below WT but had increased activity. We then transfected the RUNX1 reporter with activated Src, Runx1, or both – in several experiments Src stimulated the reporter approximately 3-fold, on average, RUNX1 4-fold, and the combination 35-fold. In the same experiments, Src + 5F led to only 6.6-fold activation, and synergy was also lost if the reporter lacked the RUNX1-binding sites. These findings, together with the observation that SHP2 directly activates Src family kinases downstream of G-CSF receptor signaling,5 raises the possibility that in myeloid cells there actually is a G-CSF to SHP2 to Src to Runx1 to Cebpa activation pathway. Consistent with this idea, we find that the 5D variant has reduced interaction with co-expressed HDAC3 relative to WT, as assessed by co-IP, whereas the 5F variant has increased interaction. We previously found that RUNX1-ER stimulates endogenous Cebpa transcription when activated in 32Dcl3 cells. As the ER segment has trans-activating activity, to evaluate RUNX1 variants we utilizing the zinc-inducible MT promoter. Preliminary analysis indicates that both WT RUNX1b and its 5D variant can induce C/EBPa protein and RNA in this context. We previously found that exogenous RUNX1-ER can rescue granulopoiesis when transduced into marrow from Runx1(flox/flox);Mx1-Cre mice subjected 4 weeks earlier to pIpC injections. We now have constructed MIGC retroviral vectors expressing RUNX1, 5F, or 5D and GFP-Cre. These, or the empty MIG or MIGC vectors, were packaged and transduced into marrow isolated from RUNX1(flox/flox) mice. Three days later, lineage-depleted, GFP+ cells were cultured in methylcellulose with IL-3, IL-6, and SCF. Compared with MIG, MIGC reduced the percent CFU-G amongst CFU-G+CFU-M, from 55+/-1% to 10+/-2%; WT RUNX1 or 5D partially rescued granulopoiesis, to 31+/- 8% or 23+/-9% respectively, whereas 5F transduction led to only 4+/-1% CFU-G. In summary, in 293T cells and potentially in myeloid cells Src kinase activates Runx1 and this may favor granulopoiesis.1. Jack et al. Blood 114:2172, 2009. 2. Zhang and Friedman. Blood 118:2266, 2011. 3. Guo et al. Blood 119:4408, 2012. 4. Huang et al. Genes Dev. 26:1587, 2012. 5. Futami et al. Blood 118:1077, 2011. Disclosures:Cantor:AMGEN: Membership on an entity's Board of Directors or advisory committees.

Neuronal activity‐dependent STAT3 localization to nucleus is dependent on Tyr‐705 and Ser‐727 phosphorylation in rat hippocampal neurons

Signal transducer and activator of transcription 3 (STAT3) dramatically increases during the first post-natal week, and supports the survival of mature hippocampal neurons. Recently, we reported that chronic elevation of excitability leads to a loss of STAT3 signal, inducing vulnerability in neurons. The loss of STAT3 signal was due to impaired Erk1/2 activation. While overnight elevation of activity attenuated STAT3 signal, brief low-frequency stimuli, which induce long-term depression, have been shown to activate STAT3. Here we investigated how STAT3 responds to depolarization in mature neurons. A brief depolarization results in the transient activation of STAT3: it induces calcium influx through L-type voltage-gated calcium channels, which triggers activation of Src family kinases. Src family kinases are required for phosphorylation of STAT3 at Tyr-705 and Ser-727. PTyr-705 is Janus kinase (JAK)-dependent, while PSer-727 is dependent on Akt, the Ser/Thr kinase. Both PTyr-705 and PSer-727 are necessary for nuclear translocation of STAT3 in these neurons. Chronic elevation of spontaneous activity by an A-type potassium blocker, 4-aminopyridine (4-AP), also induced the transient phosphorylation of STAT3, which after 4 h fell to basal levels despite the presence of 4-AP. These results suggest that phasic and chronic neuronal activation induce distinct molecular pathways, resulting in opposing regulation of STAT3 signal.

‘Clustering’ SIRPα into the Plasma Membrane Lipid Microdomains Is Required for Activated Monocytes and Macrophages to Mediate Effective Cell Surface Interactions with CD47

SIRPα, an ITIMs-containing signaling receptor, negatively regulates leukocyte responses through extracellular interactions with CD47. However, the dynamics of SIRPα-CD47 interactions on the cell surface and the governing mechanisms remain unclear. Here we report that while the purified SIRPα binds to CD47 and that SIRPα is expressed on monocytes and monocytic THP-1 or U937, these SIRPα are ineffective to mediate cell binding to immobilized CD47. However, cell binding to CD47 is significantly enhanced when monocytes transmigrating across endothelia, or being differentiated into macrophages. Cell surface labeling reveals SIRPα to be diffused on naïve monocytes but highly clustered on transmigrated monocytes and macrophages. Protein crosslink and equilibrium centrifugation confirm that SIRPα in the latter cells forms oligomerized complexes resulting in increased avidity for CD47 binding. Furthermore, formation of SIRPα complexes/clusters requires the plasma membrane ‘lipid rafts’ and the activity of Src family kinase during macrophage differentiation. These results together suggest that ‘clustering’ SIRPα into plasma membrane microdomains is essential for activated monocytes and macrophages to effectively interact with CD47 and initiate intracellular signaling.

Abstract B007: c-Src and PRC2 activity in ErbB2-driven mammary tumorigenesis and acquired resistance to ErbB2-targeted therapy

Abstract Elevated expression and activity of the non-receptor tyrosine kinase c-Src are associated with a poor prognosis in breast cancer (1). In this context c-Src, which has not been found to be mutated in breast cancer, is thought to be activated primarily through oncogenic signaling by upstream receptors. This frequently occurs in tumors driven by the overexpression of ErbB2 (2), a receptor tyrosine kinase (RTK) amplified in approximately 20-30% of all breast cancer cases (3). To investigate the mechanisms by which c-Src co-operates with activated RTK signaling to promote mammary tumorigenesis, we have developed transgenic mouse models allowing conditional c-Src ablation specifically in the mammary epithelium. In contrast to germline knockout of c-Src (4), mammary epithelial-specific deletion of c-Src does not affect ductal outgrowth. However, mammary epithelial c-Src knockout significantly delays tumorigenesis driven by ErbB2 or PyV mT, a viral oncogene that acts as an RTK mimic. The tumors that eventually develop exhibit an inflammatory and necrotic phenotype with severely impaired growth compared to wild-type controls. This phenotype correlates with alterations in the expression and activity of key metabolic regulators and with activation of the retinoblastoma tumor suppressor pathway. The expression of a known target of the Rb-E2F1 pathway, the lysine methyltransferase EZH2 (5), is also dependent on c-Src activity in transgenic mouse tumours and in human ErbB2-amplified breast cancer cell lines. A component of the Polycomb repressor complex 2 (PRC2) that di/tri-methylates histone H3 on lysine 27 (H3K27me2/me3), EZH2 is also a crucial promoter of ErbB2-driven tumour progression in vivo. Previous studies of cell line models have demonstrated that activation of Src family kinases (SFKs) is associated with the acquisition of resistance to targeted therapies directed against ErbB2 (6, 7). We demonstrate here that, in addition to elevated activity of SFKs, ErbB2+ breast cancer cell lines with acquired resistance to the ErbB2 tyrosine kinase inhibitor Lapatinib also overexpress EZH2 and have significantly increased H3K27me3 compared to parental cells. Chemical inhibition or RNAi-mediated silencing of either EZH2 or c-Src significantly attenuates the growth of Lapatinib-resistant cells, indicating a possible reversal of the drug-resistant phenotype. Overall, our data indicate that control of PRC2 expression and activity by c-Src may be an important factor in the progression of ErbB2-driven breast cancer and the acquisition of resistance to ErbB2-targeted therapies. 1. Elsberger B, Fullerton R, Zino S, Jordan F, Mitchell TJ, Brunton VG, et al. Breast cancer patients' clinical outcome measures are associated with Src kinase family member expression. Br J Cancer.103:899-909. 2. Wilson GR, Cramer A, Welman A, Knox F, Swindell R, Kawakatsu H, et al. Activated c-SRC in ductal carcinoma in situ correlates with high tumour grade, high proliferation and HER2 positivity. Br J Cancer. 2006;95:1410-4. 3. Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A, McGuire WL. Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science. 1987;235:177-82. 4. Kim H, Laing M, Muller W. c-Src-null mice exhibit defects in normal mammary gland development and ERalpha signaling. Oncogene. 2005;24:5629-36. 5. Bracken AP, Pasini D, Capra M, Prosperini E, Colli E, Helin K. EZH2 is downstream of the pRB-E2F pathway, essential for proliferation and amplified in cancer. Embo J. 2003;22:5323-35. 6. Rexer BN, Ham AJL, Rinehart C, Hill S, de Matos Granja-Ingram N, Gonzalez-Angulo AM, et al. Phosphoproteomic mass spectrometry profiling links Src family kinases to escape from HER2 tyrosine kinase inhibition. Oncogene. 2011. 7. Zhang S, Huang WC, Li P, Guo H, Poh SB, Brady SW, et al. Combating trastuzumab resistance by targeting SRC, a common node downstream of multiple resistance pathways. Nat Med. 2011;17:461-9. Citation Format: Harvey W. Smith, Alison Hirukawa, Virginie Sanguin-Gendreau, Dongmei Zuo, William J. Muller. c-Src and PRC2 activity in ErbB2-driven mammary tumorigenesis and acquired resistance to ErbB2-targeted therapy. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B007.

Low-Density Lipoprotein Receptor Related protein-1 (LRP1)-Dependent Cell Signaling Promotes Neurotrophic Activity in Embryonic Sensory Neurons

Developing sensory neurons require neurotrophic support for survival, neurite outgrowth and myelination. The low-density lipoprotein receptor-related protein-1 (LRP1) transactivates Trk receptors and thereby functions as a putative neurotrophin. Herein, we show that LRP1 is abundantly expressed in developing dorsal root ganglia (DRG) and that LRP1-dependent cell signaling supports survival, neurite extension and receptivity to Schwann cells even in the absence of neurotrophins. Cultured embryonic DRG neurons (E15) were treated with previously characterized LRP1 ligands, LRP1-receptor binding domain of α2-macroglobulin (RBD), hemopexin domain of MMP-9 (PEX) or controls (GST) for two weeks. These structurally diverse LRP1 ligands significantly activated and sustained extracellular signal-regulated kinases (ERK1/2) 5-fold (p<0.05), increased expression of growth-associated protein-43(GAP43) 15-fold (P<0.01), and increased neurite outgrowth 20-fold (P<0.01). Primary sensory neurons treated with LRP1 ligands survived > 2 weeks in vitro, to an extent equaling NGF, a finding associated with canonical signaling mechanisms and blockade of caspase-3 cleavage. LRP1 ligand-induced survival and sprouting were blocked by co-incubation with the LRP1 antagonist, receptor associated protein (RAP), whereas RAP had no effect on NGF-induced activity. Site directed mutagenesis of the LRP1 ligand, RBD, in which Lys1370 and Lys1374 are converted to alanine to preclude LRP1 binding, were ineffective in promoting cell signaling, survival or inducing neurite extension in primary sensory neurons, confirming LRP1 specificity. Furthermore, LRP1-induced neurite sprouting was mediated by Src-family kinase (SFK) activation, suggesting transactivation of Trk receptors. Co-cultures of primary embryonic neurons and Schwann cells showed that LRP1 agonists promoted axonal receptivity to myelination to Schwann cells. Collectively, these findings identify LRP1 as a novel and perhaps essential trophic molecule for sensory neuronal survival and development.

Low-density Lipoprotein Receptor-related Protein 1 (LRP1)-dependent Cell Signaling Promotes Axonal Regeneration

Low-density lipoprotein receptors (LRPs) are present extensively on cells outside of the nervous system and classically exert roles in lipoprotein metabolism. It has been reported recently that LRP1 activation could phosphorylate the neurotrophin receptor TrkA in PC12 cells and increase neurite outgrowth from developing cerebellar granule cells. These intriguing findings led us to explore the hypothesis that LRP1 activation would activate canonical neurotrophic factor signaling in adult neurons and promote axonal regeneration after spinal cord injury. We now find that treatment of adult rat dorsal root ganglion neurons in vitro with LRP1 agonists (the receptor binding domain of α-2-macroglobulin or the hemopexin domain of matrix metalloproteinase 9) induces TrkC, Akt, and ERK activation; significantly increases neurite outgrowth (p < 0.01); and overcomes myelin inhibition (p < 0.05). These effects require Src family kinase activation, a classic LRP1-mediated Trk transactivator. Moreover, intrathecal infusions of LRP1 agonists significantly enhance sensory axonal sprouting and regeneration after spinal cord injury in rats compared with control-infused animals (p < 0.05). A significant role is established for lipoprotein receptors in sprouting and regeneration after CNS injury, identifying a novel class of therapeutic targets to explore for traumatic neurological disorders.