Background CD2-associated protein (CD2AP), a slit diaphragm-associated scaffolding protein involved in survival and regulation of the cytoskeleton in podocytes, is considered a "stabilizer" of the slit diaphragm complex that connects the slit diaphragm protein nephrin to the cytoskeleton of the cell. Tyrosine phosphorylation of slit diaphragm molecules can influence their surface expression, but it is unknown whether tyrosine phosphorylation events of CD2AP are also physiologically relevant to slit diaphragm stability. Methods We used isoelectric focusing, western blot analysis, and immunofluorescence to investigate phosphorylation of CD2AP, and phospho-CD2AP antibodies and site-directed mutagenesis to define the specific phosphorylated tyrosine residues. We used cross-species rescue experiments in Cd2ap(KD) zebrafish and in Drosophila cindr(RNAi) mutants to define the physiologic relevance of CD2AP phosphorylation of the tyrosine residues. Results We found that VEGF-A stimulation can induce a tyrosine phosphorylation response in CD2AP in podocytes, and that these phosphorylation events have an important effect on slit diaphragm protein localization and functionality in vivo. We demonstrated that tyrosine in position Y10 of the SH3-1 domain of CD2AP is indispensable for CD2AP function in vivo. We found that the binding affinity of nephrin to CD2AP is significantly enhanced in the absence of Y10; however, unexpectedly, this increased affinity leads not to stabilization but to functional impairment of the glomerular filtration barrier. Conclusions Our findings provide insight into CD2AP and its phosphorylation in the context of slit diaphragm functionality, and indicate a fine-tuned affinity balance of CD2AP and nephrin that is influenced by receptor tyrosine kinase stimulation.

The clinical potential of transplantation is often reduced by T cell-mediated alloresponses that cause graft rejection or graft-versus-host disease. Integrin-mediated adhesion between alloreactive T cells and antigen-presenting cells is essential for allorejection. The identity of the signaling events needed for the activation of integrins such as LFA-1 is poorly understood. Here, we identified a novel role of the protein tyrosine phosphatase SHP-1 in the regulation of murine LFA-1-mediated adhesion in an allograft setting. Upon alloactivation, SHP-1 activity is reduced, resulting in an increase in LFA-1 adhesion compared to that for syngeneically activated T cells. The importance of these differential activation properties was further indicated by small interfering RNA (siRNA) knockdown of SHP-1 in syngeneically and allogeneically stimulated T cells. Mechanistically, SHP-1 modulated the binding of SLP-76 to ADAP by dephosphorylation of the YDGI tyrosine motif of ADAP, a known docking site for the Src family kinase Fyn. This novel key role of SHP-1 in the regulation of LFA-1-mediated adhesion may provide a new insight into T cell-mediated alloresponses and may pave the way to the development of new immunosuppressive pharmaceutical agents.

Abstract Background T cell-mediated alloresponses may result in severe transplantation-related morbidity and mortality such as graft rejection or graft versus host disease. After the adhesive interaction of a T cell and an allogeneic antigen-presenting cell an integrin-mediated, detrimental response against tissues expressing the respective alloantigen is being initiated. So far, the molecular processes underlying signaling events between T cell receptor activation and cellular adhesion are not fully understood. The vast majority of published studies was done with various tumor-derived cell lines, thus producing controversial results depending on the cell line used. Methods To overcome these drawbacks, we used in vitro generated polyclonal primary T cells based on the priming of naïve T cells from B10.A mice (responder) with denditic cells from C57BL/6 mice (allogeneic stimulator) or from B10.A mice (syngeneic stimulator) to study the lymphocyte function-associated antigen 1 (LFA-1)-mediated adhesion. Results We identified the protein tyrosine phosphatase SHP-1 as a key regulator of LFA-1-mediated adhesion. Upon alloactivation of primary T cells LFA-1-mediated adhesion was significantly higher when compared to syngeneically stimulated cells. Furthermore, elevated adhesion of alloactivated cells was accompanied by a strong decline of SHP–1 activity. To reinforce these findings, we examined the influence of a siRNA-based knockdown of SHP-1 from syngeneically and allogeneically stimulated T cells. Most notably, transfection of syngeneically stimulated cells with SHP-1-specific siRNA resulted in a two-fold increase of adhesion, when compared to cells transfected with nonsense siRNA under identical conditions. In contrast, transient overexpression of wild–type SHP-1 in allostimulated cells strongly reduced LFA–1-mediated adhesion. Since our experiments have ascertained SHP–1 as a negative regulator of the adhesion-associated signaling cascade SLP-76 → ADAP → LFA–1, we investigated the tyrosine phosphorylation of ADAP. Western blots revealed strong tyrosine phosphorylation of ADAP in allogeneically stimulated cells, but not in syngeneically stimulated cells. Furthermore, SHP-1 binding with ADAP was significantly stronger in syngeneically stimulated cells, when compared to allogeneically stimulated cells. Besides these experiments, substrate specificity of SHP–1 to ADAP confirmed by in vitro dephosphorylation asssays with GST-SHP–1 and GST-SHP–2 fusion proteins indicate that SHP–1 modulates the binding of SLP–76 with ADAP by tyrosine dephosphorylation of ADAP. Conclusion These findings might be of clinical relevance, since the decisive role of SHP–1 in the regulation of LFA–1-mediated adhesion in alloresponses allows the development of novel target-specific immunosuppressive agents. Disclosures: No relevant conflicts of interest to declare.

In response to DNA damage, transcription is blocked by inhibition of RNA polymerase II activity. The regulation of a preexisting pool of mRNAs, therefore, plays a key role in DNA repair, cell cycle arrest, or inhibition of differentiation. THOC5 is a member of the THO complex and plays a role in the export of a subset of mRNA, which plays an important role in hematopoiesis and maintaining primitive cells. Since three serine residues in the PEST domain of THOC5 have been shown to be directly phosphorylated by ataxia-telangiectasia-mutated (ATM) kinase, we examined the THOC5-dependent mRNA export under DNA damage. We show here that DNA damage drastically decreased the cytoplasmic pool of a set of THOC5-dependent mRNAs and impaired the THOC5/mRNA complex formation. The mRNP complex formed with nonphosphorylation mutant (S307/312/314A) THOC5, but not with a C-terminal deletion mutant after DNA damage, suggesting that the C-terminal domain of THOC5, but not its phosphorylation in the PEST domain, is necessary for the regulation of the mRNA-binding potency of THOC5. The cytoplasmic THOC5-dependent mRNAs were recovered by treatment with ATM kinase-specific or p53-specific siRNA, as well as by treatment with ATM kinase inhibitor, KU55933, under DNA damage conditions, suggesting that the ATM-kinase-p53 pathway is involved in this response to the DNA damage. Furthermore, the treatment with KU55933 blocked DNA damage-induced THOC5mRNP complex dissociation, indicating that activation of ATM kinase suppresses the ability of THOC5 to bind to its target mRNAs.

Protein tyrosine phosphorylation controls a wide range of critical biological processes. Recently, we provided the first evidence that the level of tyrosine phosphorylation of the cell cycle regulator p27Kip1 plays a crucial role in its binding to cyclin dependent kinases and its subcellular localization. While Src and Bcr-Abl were shown to be responsible for tyrosine phosphorylation, no data are available on the dephosphorylation of p27Kip1 and the phosphatase involved. Considering the associated dephosphorylation as a pivotal event in the regulation of cell cycle proteins, we focused on the tyrosine phosphatase SHP-2, which is regulated in promyelocytic leukemia cells on stimulation with G-CSF. SHP-2 was thus found in association with p27Kip1 and the G-CSF receptor, and we observed a nuclear translocation of SHP-2 on G-CSF stimulation. Using a catalytically inactive form of SHP-2 and siRNA directed against SHP-2, we could demonstrate the involvement of SHP-2 in tyrosine dephosphorylation of p27Kip1. Moreover, SHP-2 was strongly activated on G-CSF stimulation and specifically dephosphorylated p27Kip1 in vitro. Most importantly, we could illustrate that SHP-2 modulates p27Kip1 stability and contributes to p27Kip1-mediated cell cycle progression. Taken together, our results demonstrate that SHP-2 is a key regulator of p27Kip1 tyrosine phosphorylation.

Receptor tyrosine kinase-mediated signaling is tightly regulated by a number of cytoplasmic signaling molecules. In this report, we show that Bcr–Abl transformed chronic myelogenous leukemia (CML) cell lines, K562 and Meg-01, express the receptor for nerve growth factor (NGF), TrkA, on the cell surface; however, the NGF-mediated signal is not particularly strong. Treatment with imatinib, a potent inhibitor of Bcr–Abl tyrosine kinase, downmodulates phosphorylation of downstream molecules. Upon stimulation with NGF, Erk and Akt are phosphorylated to a much greater degree in imatinib-treated cells than in untreated cells. Knockdown of expression of Bcr–Abl using small interfering RNA technique also enhanced NGF-mediated Akt phosphorylation, indicating that Bcr–Abl kinase modifies NGF signaling directly. Imatinib treatment also enhanced NGF signaling in rat adrenal pheochromocytoma cell line PC12 that expresses TrkA and c-Abl, suggesting that it is not only restoration of responsiveness to NGF after blocking oncoprotein activity, but also c-Abl tyrosine kinase per se may be a negative regulator of growth factor signaling. Furthermore, inhibition of Abl tyrosine kinase enhanced clearance of surface TrkA after NGF treatment and simultaneously enhanced NGF-mediated signaling, suggesting that as in neuronal cells ‘signaling endosomes’ are formed in hematopoietic cells. To examine the role of TrkA in CML cells, we studied cell growth or colony formation in the presence or absence of imatinib with or without NGF. We found that NGF treatment induces cell survival in imatinib-treated CML cell lines, as well as colony formation of primary CD34+ CML cells, strongly suggesting that NGF/TrkA signaling contributes to aberrant signaling in CML.

Tyrosine phosphorylation of the cell cycle regulator p27Kip1 plays a crucial role in its binding to cyclin dependent kinases and its subcellular localization. While Src and Bcr-Abl were shown to be responsible for tyrosine phosphorylation, no data are available on the dephosphorylation of p27Kip1 and the phosphatase involved. Considering the associated dephosphorylation as a pivotal event in the regulation of cell cycle proteins, we focused on the tyrosine phosphatase SHP-2, which is regulated in promyelocytic leukemia cells on G-CSF stimulation. SHP-2 was thus found in association with p27Kip1 and the G-CSF receptor, and we observed a nuclear translocation of SHP-2 on G-CSF stimulation. Using a catalytically inactive form of SHP-2 and siRNA directed against SHP-2, we could demonstrate the involvement of SHP-2 in tyrosine dephosphorylation of p27Kip1. Moreover, SHP-2 was strongly activated on G-CSF stimulation and specifically dephosphorylated p27Kip1 in vitro. Most importantly, we could illustrate that SHP-2 modulates p27Kip1 stability and contributes to p27Kip1-mediated cell cycle progression. Taken together, our results demonstrate that SHP-2 is a key regulator of p27Kip1 tyrosine phosphorylation.

Defects in podocyte signaling are the basis of many inherited glomerular diseases leading to glomerulosclerosis. CD2-associated protein (CD2AP) is highly expressed in podocytes and is considered to play an important role in the maintenance of the glomerular slit diaphragm. Mice deficient for CD2AP (CD2AP-/-) appear normal at birth but develop a rapid onset nephrotic syndrome at 3 weeks of age. We demonstrate that impaired intracellular signaling with subsequent podocyte damage is the reason for this delayed podocyte injury in CD2AP-/- mice. We document that CD2AP deficiency in podocytes leads to diminished signal initiation and termination of signaling pathways mediated by receptor tyrosine kinases (RTKs). In addition, we demonstrate that CIN85, a paralog of CD2AP, is involved in termination of RTK signaling in podocytes. CIN85 protein expression is increased in CD2AP-/- podocytes in vitro. Stimulation of CD2AP-/- podocytes with various growth factors, including insulin-like growth factor 1, vascular endothelial growth factor, and fibroblast growth factor, resulted in a significantly decreased phosphatidylinositol 3-kinase/AKT and ERK signaling response. Moreover, increased CIN85 protein is detectable in podocytes in diseased CD2AP-/- mice, leading to decreased base-line activation of ERK and decreased phosphorylation after growth factor stimulation in vivo. Because repression of CIN85 protein leads to a restored RTK signaling response, our results support an important role of CD2AP/CIN85 protein balance in the normal signaling response of podocytes.

We have investigated the role of tyrosine phosphorylation of the cyclin-dependent kinase (cdk) inhibitor p27Kip1 using the acute promyelocytic leukemia cell line NB4 together with granulocyte colony-stimulating factor (G-CSF). Short-term G-CSF stimulation resulted in a rapid tyrosine dephosphorylation of p27Kip1 accompanied by a change in its binding preferences to cdks. On G-CSF stimulation, p27Kip1 dissociated from cdk4 and associated with cdk2. Binding assays with recombinant p27Kip1 confirmed that tyrosine-phosphorylated p27Kip1 preferentially bound to cdk4, whereas unphosphorylated protein preferentially associated with cdk2. In addition, studies with p27Kip1 point mutations revealed a decisive role of Tyr88 and Tyr89 in binding to cdk4. Furthermore, phosphorylation of Tyr88 and Tyr89 was accompanied by strong nuclear translocation of p27Kip1. Taken together, this report provides the first evidence that tyrosine phosphorylation of p27Kip1 plays a crucial role in binding to cdks and its subcellular localization. Moreover, both effects are mediated by application of G-CSF.

The response to initial glucocorticoid therapy in childhood acute lymphoblastic leukaemia (ALL) reliably predicts the response to multiagent chemotherapy. Patients resistant to glucocorticoids (prednisone poor responders (PPR)) have a poorer event-free survival compared to glucocorticoid-sensitive patients (prednisone good responders (PGR)). A case–control study was performed to investigate differential protein expression in leukaemic blasts from PGR and PPR childhood ALL patients. Two-dimensional gel electrophoresis (2-DE) was used for an unsupervised screening and surface enhanced laser desorption/ionisation-time of flight mass spectrometry (SELDI-TOF MS) for the characterisation of protein spots. In difference maps of average gels for the proteomes of each responder group, differentially expressed proteins were identified after tryptic digestion and spotting onto H4-SELDI-TOF-MS chips. Proteins overexpressed in PPR were Catalase, RING finger protein 22 alpha, Valosin-containing protein (VCP) and a G-protein-coupled receptor. Proteins overexpressed in PGR were protein kinase C and malate dehydrogenase. Valosin-containing protein was chosen for validation and quantification by Western blot analysis in a second case–control group of ALL patients. In this second independent cohort, median VCP expression (P 25 –P 75 ) was 0.15 (0.11–0.28) in PGR and 0.34 (0.14–0.99) in PPR patients ( P =0.04). We conclude that high VCP expression is associated with poor prednisone response in childhood ALL patients.

word count: 148; total text word count: 4,845 This work was funded by grant SFB566 of the Deutsche Forschungsgemeinschaft (DFG). Contributions of authors: CK: conception, designed and performed research, wrote paper; MD: performed research, wrote paper; AKardinal: performed research; AlexandraK: analytical tools; DTB: reviewed and discussed research; TT: designed and discussed research; KW: conception and discussion Blood First Edition Paper, prepublished online September 29, 2005; DOI 10.1182/blood-2005-05-1771 Copyright © 2005 American Society of Hematology For personal use only. on March 31, 2017. by guest www.bloodjournal.org From

The anti-viral type I interferon (IFN) response is initiated by the immediate induction of IFN beta, which is mainly controlled by the IFN-regulatory factor-3 (IRF-3). The signaling pathways mediating viral IRF-3 activation are only poorly defined. We show that the Rho GTPase Rac1 is activated upon virus infection and controls IRF-3 phosphorylation and activity. Inhibition of Rac1 leads to reduced IFN beta promoter activity and to enhanced virus production. As a downstream mediator of Rac signaling towards IRF-3, we have identified the kinase p21-activated kinase (PAK1). Furthermore, both Rac1 and PAK1 regulate the recently described IRF-3 activators, I kappa B kinase- and TANK-binding kinase-1, establishing a first canonical virus-induced IRF-3 activating pathway.

Early reduction of leukaemic cells by chemotherapy is a strong predictor for treatment outcome in childhood acute lymphoblastic leukaemia (ALL). In ALL–(Berlin–Frankfurt–Münster) trials, early treatment response is assessed by the in vivo response to glucocorticoids (prednisone response, PR), the molecular background of which is unknown. The intracellular effects of glucocorticoids (GCs) are mediated by the glucocorticoid receptor (GR). In the absence of GC, the inactive GR resides within a multiprotein complex, consisting predominantly of the chaperone protein hsp90 (heat-shock protein 90). Until now, studies targeting GC resistance mainly focused on GR disorders and alterations of genes known to be associated with drug resistance. In addition, the GR multiprotein complex was associated with GC resistance in in vitro studies. We performed a case–control study for PR to investigate the association of in vivo GC resistance and hsp90 expression in childhood ALL. Hsp90 expression was assessed using a real-time PCR approach (Taqman technology) and Western blot technology. In this setting, we found no association of in vivo GC resistance and hsp90 expression. Therefore, we conclude that the expression of hsp90, the major component of the GR activating complex, is of minor importance for the in vivo GC resistance in childhood ALL.

Herpesvirus saimiri codes for a tyrosine kinase interacting protein (Tip) that interacts with both the SH3 domain and the kinase domain of the T-cell-specific tyrosine kinase Lck via two separate motifs. The activation of Lck by Tip is considered as a key event in the transformation of human T-lymphocytes during herpesviral infection. We investigated the interaction of proline-rich Tip peptides with the LckSH3 domain starting with the structural characterization of the unbound interaction partners. The solution structure of the LckSH3 was determined by heteronuclear multidimensional nuclear magnetic resonance (NMR) spectroscopy using 44 residual dipolar couplings in addition to the conventional experimental restraints. Circular dichroism spectroscopy proved that the polyproline helix of Tip is already formed prior to SH3 binding and is conformationally stable. NMR titration experiments point out three major regions of the Tip-Lck interaction comprising the RT loop, the n-src loop, and a helical turn preceding the last strand of the beta-sheet. Further changes of the chemical shifts were observed for the N- and C-terminal beta-strands of the SH3 domain, indicating additional contacts outside the proline-rich segment or subtle structural rearrangements transmitted from the binding site of the proline helix. Fluorescence spectroscopy shows that Tip binds to the SH3 domains of several Src kinases (Lck, Hck, Lyn, Src, Fyn, Yes), exhibiting the highest affinities for Lyn, Hck, and Lck.

Pseudo-proline building blocks exert a dual functionality in enhancing and stabilizing the relevant polyproline II (PPII) conformation and increasing and optimizing van der Waals contacts and hydrogen bonding to the receptor mols. thus modifying affinity and specificity. They are highly useful in studying ligand recognition mediated by Src homol. 3 domains essential in cellular regulation and protein-protein interactions. The 2-C substituents promote the induction of the required PPII helix and allow for optimal complementation of the SH3 topog. [on SciFinder (R)]

Chronic myelogenous leukemia (CML) is commonly characterized by the presence of the p210(Bcr-Abl) oncoprotein. Many downstream effectors of Bcr-Abl have been described, including activation of the Grb2-SoS-Ras-MAP kinase (Erk) pathway. The precise contributions of these signal-transduction proteins in CML blast cells in human patients are not yet well defined. To gain further insight into the importance of Grb2 for CML, peptides that disrupt Grb2-SoS complexes were tested. These high-affinity Grb2-binding peptides (HAGBPs) can autonomously shuttle into cells and function by binding to the N-terminal SH3 domain of Grb2. The HAGBPs were analyzed for their effects on Bcr-Abl-expressing cell lines and freshly isolated CML blast cells from patients. They induced a dramatic decrease in the proliferation of CML cell lines. This was not observed with point-mutated control peptides with abolished Grb2SH3(N) binding. As expected, Grb2-SoS complexes were greatly diminished in the HAGBP-treated cells, and MAP kinase activity was significantly reduced as determined by an activation-specific phospho-MAPK antibody. Furthermore, cell fractions that are enriched for blast cells from CML patients with active disease were also incubated with the Grb2 blocker peptides. The HAGBPs led to a significant proliferation reduction of these cells in the majority of the isolates, but not in all patients' cells. These results show that, in addition to the direct targeting of Bcr-Abl, selective inhibition of Grb2 protein complexes may be a therapeutic option for a significant number of CML patients.

Hepatocyte growth factor (HGF; scatter factor) is a multipotent growth and morphogenesis factor implicated in cell migration, developmental processes, and proliferation. Recent work has shown that HGF activates the receptor tyrosine kinase c-Met which phosphorylates the large docking protein Gab1, leading to the recruitment of several signaling molecules, including the Crk family adapter proteins c-Crk and Crk-like (CRKL). HEK293 (human embryonic kidney) cells treated with HGF show reduced cell adhesion. In the present study it is documented that the HGF-induced adhesion loss is blocked by disruption of the Crk/CRKL signaling through the forced expression of a dominant negative Crk/CRKLSH3(1) binding fragment (CBR) of the guanine releasing protein C3G as well as by incubation with cell-penetrating Crk/CRKLSH3(1) domain blocker peptides. CBR expression also inhibits HGF-induced activation of N-terminal c-Jun kinase (JNK) and HGF-induced activation of the small GTPase Rac. We conclude that Crk family adapters are essential mediators of HGF-induced signaling events.

Pseudoprolines (ΨPro) have been developed as tools for inducing bioactive conformations that allow for optimal spatial complementation in protein-protein interactions. This dual function of ΨPro was explored for tuning proline-rich peptides as potent ligands for SH3 domains.