A Global Analysis of the Receptor Tyrosine Kinase-Protein Phosphatase Interactome.

Receptor tyrosine kinases (RTKs) and protein phosphatases comprise protein families that play crucial roles in cell signaling. We used two protein-protein interaction (PPI) approaches, the membrane yeast two-hybrid (MYTH) and the mammalian membrane two-hybrid (MaMTH), to map the PPIs between human RTKs and phosphatases. The resulting RTK-phosphatase interactome reveals a considerable number of previously unidentified interactions and suggests specific roles for different phosphatase families. Additionally, the differential PPIs of some protein tyrosine phosphatases (PTPs) and their mutants suggest diverse mechanisms of these PTPs in the regulation of RTK signaling. We further found that PTPRH and PTPRB directly dephosphorylate EGFR and repress its downstream signaling. By contrast, PTPRA plays a dual role in EGFR signaling: besides facilitating EGFR dephosphorylation, it enhances downstream ERK signaling by activating SRC. This comprehensive RTK-phosphatase interactome study provides a broad and deep view of RTK signaling.

A ERK/RSK-mediated negative feedback loop regulates M-CSF-evoked PI3K/AKT activation in macrophages.

Activation of the RAS/ERK and its downstream signaling components is essential for growth factor-induced cell survival, proliferation, and differentiation. The Src homology-2 domain containing protein tyrosine phosphatase 2 (SHP2), encoded by protein tyrosine phosphatase, non-receptor type 11 ( Ptpn11), is a positive mediator required for most, if not all, receptor tyrosine kinase-evoked RAS/ERK activation, but differentially regulates the PI3K/AKT signaling cascade in various cellular contexts. The precise mechanisms underlying the differential effects of SHP2 deficiency on the PI3K pathway remain unclear. We found that mice with myelomonocytic cell-specific [ Tg(LysM-Cre); Ptpn11fl/fl mice] Ptpn11 deficiency exhibit mild osteopetrosis. SHP2-deficient bone marrow macrophages (BMMs) showed decreased proliferation in response to M-CSF and decreased osteoclast generation. M-CSF-evoked ERK1/2 activation was decreased, whereas AKT activation was enhanced in SHP2-deficient BMMs. ERK1/2, via its downstream target RSK2, mediates this negative feedback by negatively regulating phosphorylation of M-CSF receptor at Tyr721 and, consequently, its binding to p85 subunit of PI3K and PI3K activation. Pharmacologic inhibition of RSK or ERK phenotypically mimics the signaling defects observed in SHP2-deficient BMMs. Furthermore, this increase in PI3K/AKT activation enables BMM survival in the setting of SHP2 deficiency.-Wang, L., Iorio, C., Yan, K., Yang, H., Takeshita, S., Kang, S., Neel, B.G., Yang, W. An ERK/RSK-mediated negative feedback loop regulates M-CSF-evoked PI3K/AKT activation in macrophages.

Substrate specificity of protein tyrosine phosphatases 1B, RPTPα, SHP-1, and SHP-2.

We determined the substrate specificities of the protein tyrosine phosphatases (PTPs) PTP1B, RPTPα, SHP-1, and SHP-2 by on-bead screening of combinatorial peptide libraries and solution-phase kinetic analysis of individually synthesized phosphotyrosyl (pY) peptides. These PTPs exhibit different levels of sequence specificity and catalytic efficiency. The catalytic domain of RPTPα has very weak sequence specificity and is approximately 2 orders of magnitude less active than the other three PTPs. The PTP1B catalytic domain has modest preference for acidic residues on both sides of pY, is highly active toward multiply phosphorylated peptides, but disfavors basic residues at any position, a Gly at the pY-1 position, or a Pro at the pY+1 position. By contrast, SHP-1 and SHP-2 share similar but much narrower substrate specificities, with a strong preference for acidic and aromatic hydrophobic amino acids on both sides of the pY residue. An efficient SHP-1/2 substrate generally contains two or more acidic residues on the N-terminal side and one or more acidic residues on the C-terminal side of pY but no basic residues. Subtle differences exist between SHP-1 and SHP-2 in that SHP-1 has a stronger preference for acidic residues at the pY-1 and pY+1 positions and the two SHPs prefer acidic residues at different positions N-terminal to pY. A survey of the known protein substrates of PTP1B, SHP-1, and SHP-2 shows an excellent agreement between the in vivo dephosphorylation pattern and the in vitro specificity profiles derived from library screening. These results suggest that different PTPs have distinct sequence specificity profiles and the intrinsic activity/specificity of the PTP domain is an important determinant of the enzyme's in vivo substrate specificity.

Silencing the G-protein coupled receptor 3-salt inducible kinase 2 pathway promotes human ß cell proliferation.

Loss of pancreatic ß cells is the hallmark of type 1 diabetes, for which provision of insulin is the standard of care. While regenerative and stem cell therapies hold the promise of generating single-source or host-matched tissue to obviate immune-mediated complications, these will still require surgical intervention and immunosuppression. Here we report the development of a high-throughput RNAi screening approach to identify upstream pathways that regulate adult human ß cell quiescence and demonstrate in a screen of the GPCRome that silencing G-protein coupled receptor 3 (GPR3) leads to human pancreatic ß cell proliferation. Loss of GPR3 leads to activation of Salt Inducible Kinase 2 (SIK2), which is necessary and sufficient to drive cell cycle entry, increase ß cell mass, and enhance insulin secretion in mice. Taken together, our data show that targeting the GPR3-SIK2 pathway is a potential strategy to stimulate the regeneration of ß cells.

Murine Pif1 interacts with telomerase and is dispensable for telomere function in vivo.

Pif1 is a 5'-to-3' DNA helicase critical to DNA replication and telomere length maintenance in the budding yeast Saccharomyces cerevisiae. ScPif1 is a negative regulator of telomeric repeat synthesis by telomerase, and recombinant ScPif1 promotes the dissociation of the telomerase RNA template from telomeric DNA in vitro. In order to dissect the role of mPif1 in mammals, we cloned and disrupted the mPif1 gene. In wild-type animals, mPif1 expression was detected only in embryonic and hematopoietic lineages. mPif1(-/-) mice were viable at expected frequencies, displayed no visible abnormalities, and showed no reproducible alteration in telomere length in two different null backgrounds, even after several generations. Spectral karyotyping of mPif1(-/-) fibroblasts and splenocytes revealed no significant change in chromosomal rearrangements. Furthermore, induction of apoptosis or DNA damage revealed no differences in cell viability compared to what was found for wild-type fibroblasts and splenocytes. Despite a novel association of mPif1 with telomerase, mPif1 did not affect the elongation activity of telomerase in vitro. Thus, in contrast to what occurs with ScPif1, murine telomere homeostasis or genetic stability does not depend on mPif1, perhaps due to fundamental differences in the regulation of telomerase and/or telomere length between mice and yeast or due to genetic redundancy with other DNA helicases.

Prompt and sustained response of a steroid-refractory autoimmune hemolytic anemia to a rituximab-based therapy in a chronic lymphocytic leukemia patient.

INTRODUCTION: Autoimmune hemolytic anemia (AIHA) is a rare and potentially life-threatening event which may complicate the course of chronic lymphocytic leukemia (CLL) at any time and steroid-refractory AIHA of CLL poses a therapeutic challenge for physicians. Here, we report the safety and efficacy of a rituximab-containing regimen in a CLL patient with steroid- and IVIg-refractory AIHA. CASE REPORT: A 57-year- old man affected by CLL, presented with fatigue, dyspnoea, tachycardia and jaundice. His physical examination revealed overt jaundice, hepato- and splenomegaly, and enlargement of lymph nodes in all superficial sites. The blood chemistry showed severe anemia (Hb value 3.9 g/dL), high white blood cell count (89 x 10(9)/L), altered hemolysis markers and direct antiglobulin test (DAT) was positive for both complement and IgG. The patient failed to respond to both a 4-day course of high-dose dexamethasone IV (40 mg/day) and intravenous immunoglobulin (IVIg) (1 g/kg/day x 2 days). Thus, a schedule containing rituximab (375 mg/m(2) day +1), cyclophosphamide (750 mg/m(2) day +2) and prednisone (60 mg/m(2) from day +1 to day +7) (R-CP) were administered. Four cycles, repeated every 4 weeks, were administered. After 4 days from the infusion of this schedule, the patient showed a marked reduction of the lymphocytosis, and the hemoglobin level started to increase. No rituximab-related side effects were recorded. At the end of treatment DAT became negative and patient achieved a nodular Partial Remission (nPR). CONCLUSION: Our data showed the safety and efficacy of a rituximab-containing regimen in a life-threatening CLL-related AIHA, refractory to steroid and IVIg therapy. This schedule has allowed the patient to obtain a prompt and dramatic rise in hemoglobin level and a response to both AIHA and CLL.

Differentiation of Normal and Radioresistant Prostate Cancer Xenografts Using Magnetization Transfer-Prepared MRI.

The ability of MRI to differentiate between normal and radioresistant cancer was investigated in prostate tumour xenografts in mice. Specifically, the process of magnetization exchange between water and other molecules was studied. It was found that magnetization transfer from semisolid macromolecules (MT) and chemical exchange saturation transfer (CEST) combined were significantly different between groups (p < 0.01). Further, the T2 relaxation of the semisolid macromolecular pool (T2,B), a parameter specific to MT, was found to be significantly different (p < 0.01). Also significantly different were the rNOE contributions associated with methine groups at -0.9 ppm with a saturation B1 of 0.5 µT (p < 0.01) and with other aliphatic groups at -3.3 ppm with 0.5 and 2 µT (both p < 0.05). Independently, using a live-cell metabolic assay, normal cells were found to have a greater metabolic rate than radioresistant ones. Thus, MRI provides a novel, in vivo method to quantify the metabolic rate of tumours and predict their radiosensitivity.

PTP1B controls non-mitochondrial oxygen consumption by regulating RNF213 to promote tumour survival during hypoxia.

Tumours exist in a hypoxic microenvironment and must limit excessive oxygen consumption. Hypoxia-inducible factor (HIF) controls mitochondrial oxygen consumption, but how/if tumours regulate non-mitochondrial oxygen consumption (NMOC) is unknown. Protein-tyrosine phosphatase-1B (PTP1B) is required for Her2/Neu-driven breast cancer (BC) in mice, although the underlying mechanism and human relevance remain unclear. We found that PTP1B-deficient HER2(+) xenografts have increased hypoxia, necrosis and impaired growth. In vitro, PTP1B deficiency sensitizes HER2(+) BC lines to hypoxia by increasing NMOC by α-KG-dependent dioxygenases (α-KGDDs). The moyamoya disease gene product RNF213, an E3 ligase, is negatively regulated by PTP1B in HER2(+) BC cells. RNF213 knockdown reverses the effects of PTP1B deficiency on α-KGDDs, NMOC and hypoxia-induced death of HER2(+) BC cells, and partially restores tumorigenicity. We conclude that PTP1B acts via RNF213 to suppress α-KGDD activity and NMOC. This PTP1B/RNF213/α-KGDD pathway is critical for survival of HER2(+) BC, and possibly other malignancies, in the hypoxic tumour microenvironment.

Measles virus replication in lymphatic cells and organs of CD150 (SLAM) transgenic mice.

A transgenic mouse containing the complete human SLAM (hSLAM/CD150) gene, including its endogenous promoter for transcription, was generated by using human genomic DNA cloned into a bacterial artificial chromosome. hSLAM, the primary receptor for measles viruses (MV), was expressed on activated B, T, and dendritic cells with an expression profile equivalent to that of humans. We demonstrated that hSLAM(+) cells obtained from the transgenic mouse, including activated B, T, and dendritic cells, were susceptible to MV infection in a receptor-dependent manner. Evidence was provided for transient infection in the nasal lymph nodes of hSLAM(+) mice after intranasal inoculation. Virus was rapidly cleared without signs of secondary replication. To improve the efficiency of MV production, the hSLAM(+) mice were bred with mice having a Stat1-deficient background. These mice were more susceptible to MV infection and produced more virus particles. After intranasal and intraperitoneal inoculation of these mice with MV, infections of the thymus, spleen, nasal, mesenteric, and leg lymph nodes were detected. Upon necropsy, enlarged lymph nodes and spleen were apparent. Flow cytometric analysis showed that abnormally large numbers of mature neutrophils and natural killer cells caused the splenomegaly. The hSLAM transgenic mouse constitutes an improved rodent model for studying the interaction of MV with immune cells that more accurately reflects the infection pattern found in humans.

Mechanism of CD150 (SLAM) down regulation from the host cell surface by measles virus hemagglutinin protein.

Measles virus has been reported to enter host cells via either of two cellular receptors, CD46 and CD150 (SLAM). CD46 is found on most cells of higher primates, while SLAM is expressed on activated B, T, and dendritic cells and is an important regulatory molecule of the immune system. Previous reports have shown that measles virus can down regulate expression of its two cellular receptors on the host cell surface during infection. In this study, the process of down regulation of SLAM by measles virus was investigated. We demonstrated that expression of the hemagglutinin (H) protein of measles virus was sufficient for down regulation. Our studies provided evidence that interactions between H and SLAM in the endoplasmic reticulum (ER) can promote the down regulation of SLAM but not CD46. In addition, we demonstrated that interactions between H and SLAM at the host cell surface can also contribute to SLAM down regulation. These results indicate that two mechanisms involving either intracellular interactions between H and SLAM in the ER or receptor-mediated binding to H at the surfaces of host cells can lead to the down regulation of SLAM during measles virus infection.