Tyrosine Phosphorylation of CD2AP Affects Stability of the Slit Diaphragm Complex.

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 Cd2apKD zebrafish and in Drosophila cindrRNAi 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.

SHP-1 Acts as a Key Regulator of Alloresponses by Modulating LFA-1-Mediated Adhesion in Primary Murine T Cells.

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.

An ataxia-telangiectasia-mutated (ATM) kinase mediated response to DNA damage down-regulates the mRNA-binding potential of THOC5.

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.

Tyrosine phosphatase SHP-2 is a regulator of p27(Kip1) tyrosine phosphorylation.

Tyrosine phosphorylation of the cell cycle regulator p27(Kip1) 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 p27(Kip1) 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 p27(Kip1) 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 p27(Kip1). Moreover, SHP-2 was strongly activated on G-CSF stimulation and specifically dephosphorylated p27(Kip1) in vitro. Most importantly, we could illustrate that SHP-2 modulates p27(Kip1) stability and contributes to p27(Kip1)-mediated cell cycle progression. Taken together, our results demonstrate that SHP-2 is a key regulator of p27(Kip1) tyrosine phosphorylation.

CD2AP/CIN85 balance determines receptor tyrosine kinase signaling response in podocytes.

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.

Tyrosine phosphorylation modulates binding preference to cyclin-dependent kinases and subcellular localization of p27Kip1 in the acute promyelocytic leukemia cell line NB4.

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.

Rac1 and PAK1 are upstream of IKK-epsilon and TBK-1 in the viral activation of interferon regulatory factor-3.

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.

Structural investigation of the binding of a herpesviral protein to the SH3 domain of tyrosine kinase Lck.

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.

Targeting Molecular Recognition: Exploring the Dual Role of Functional Pseudoprolines in the Design of SH3 Ligands.

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.