SH2-containing Inositol Research Articles

Drugs targeting SHIP2 demonstrate potent antiproliferative effects irrespective of SHIP2 inhibition

The SH2-containing inositol 5′-phosphatase SHIP2 plays a crucial role in negative regulation of the PI3K/AKT signaling pathway. Putative small molecule inhibitors of SHIP2, AS1949490 and K149 have been reported to elicit a range of beneficial effects in treating or preventing obesity as well as killing cancer cells. However, whether these effects are direct results of SHIP2 inhibition has not been carefully assessed, e.g., in the absence of expression of the protein. Here, we show that these inhibitors alter the PI3K/AKT signaling pathway irrespective of SHIP2 protein expression. Moreover, we found that AS1949490 and K149 alter cell growth in normal and cancer cells lacking both SHIP1 and SHIP2. Overall, our data provide evidence that the antiproliferative effects of AS1949490 and K149 cannot be attributed to SHIP1/2 inhibition.

Enzyme Is the Name-Adapter Is the Game.

Signaling proteins in eukaryotes usually comprise a catalytic domain coupled to one or several interaction domains, such as SH2 and SH3 domains. An additional class of proteins critically involved in cellular communication are adapter or scaffold proteins, which fulfill their purely non-enzymatic functions by organizing protein-protein interactions. Intriguingly, certain signaling enzymes, e.g., kinases and phosphatases, have been demonstrated to promote particular cellular functions by means of their interaction domains only. In this review, we will refer to such a function as "the adapter function of an enzyme". Though many stories can be told, we will concentrate on several proteins executing critical adapter functions in cells of the immune system, such as Bruton´s tyrosine kinase (BTK), phosphatidylinositol 3-kinase (PI3K), and SH2-containing inositol phosphatase 1 (SHIP1), as well as in cancer cells, such as proteins of the rat sarcoma/extracellular signal-regulated kinase (RAS/ERK) mitogen-activated protein kinase (MAPK) pathway. We will also discuss how these adaptor functions of enzymes determine or even undermine the efficacy of targeted therapy compounds, such as ATP-competitive kinase inhibitors. Thereby, we are highlighting the need to develop pharmacological approaches, such as proteolysis-targeting chimeras (PROTACs), that eliminate the entire protein, and thus both enzymatic and adapter functions of the signaling protein. We also review how genetic knock-out and knock-in approaches can be leveraged to identify adaptor functions of signaling proteins.

SHIP inhibition mediates select TREM2-induced microglial functions

Microglia play a pivotal role in the pathology of Alzheimer's Disease (AD), with the Triggering Receptor Expressed on Myeloid cells 2 (TREM2) central to their neuroprotective functions. The R47H variant of TREM2 has emerged as a significant genetic risk factor for AD, leading to a loss-of-function phenotype in mouse AD models. This study elucidates the roles of TREM2 in human microglia-like HMC3 cells and the regulation of these functions by SH2-containing inositol-5′-phosphatase 1 (SHIP1). Using stable cell lines expressing wild-type TREM2, the R47H variant, and TREM2-deficient lines, we found that functional TREM2 is essential for the phagocytosis of Aβ, lysosomal capacity, and mitochondrial activity. Notably, the R47H variant displayed increased phagocytic activity towards apoptotic neurons. Introducing SHIP1, known to modulate TREM2 signaling in other cells, revealed its role as a negative regulator of these TREM2-mediated functions. Moreover, pharmacological inhibition of both SHIP1 and its isoform SHIP2 amplified Aβ phagocytosis and lysosomal capacity, independently of TREM2 or SHIP1 expression, suggesting a potential regulatory role for SHIP2 in these functions. The absence of TREM2, combined with the presence of both SHIP isoforms, suppressed mitochondrial activity. However, pan-SHIP1/2 inhibition enhanced mitochondrial function in these cells. In summary, our findings offer a deeper understanding of the relationship between TREM2 variants and SHIP1 in microglial functions, and emphasize the therapeutic potential of targeting the TREM2 and SHIP1 pathways in microglia for neurodegenerative diseases.

Mechanism of Notoginseng Radix et Rhizoma in regulating PI3K/Akt/mTORC1-mitochondrial energy metabolism to treat chronic renal failure in rats with blood stasis syndrome

This study observed the effects of Notoginseng Radix et Rhizoma on the phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)/mammalian target of rapamycin complex 1(mTORC1) signaling pathway and mitochondrial energy metabolism in the rat model of adriamycin-induced renal fibrosis with blood stasis syndrome to explore the mechanism of Notoginseng Radix et Rhizoma in protecting the kidney. Thirty male rats with adriamycin-induced renal fibrosis were randomized into model, low-, medium-, and high-dose Notoginseng Radix et Rhizoma, and positive control groups(n=6). Six clean SD male rats were selected into the normal group. The normal group and model group were administrated with normal saline, and other groups with corresponding drugs. After 8 weeks of treatment, the renal function, renal pathology, adenosine triphosphate(ATP) levels, Na~+-K~+-ATPase and Ca~(2+)-Mg~(2+)-ATPase activities, and the protein levels of ATP5B, mTORC1, 70 kDa ribosomal protein S6 kinase(P70S6K), P85, Akt, p-Akt, and SH2-containing inositol phosphatase(SHIP2) in the renal tissue were determined. Compared with the normal group, the model group showed elevated levels of blood urea nitrogen(BUN) and serum creatinine(SCr)(P<0.01). Compared with the model group, Notoginseng Radix et Rhizoma and the positive control lowered the levels of BUN and SCr, which were significant in the medium-and high-dose Noto-ginseng Radix et Rhizoma groups and the positive control group(P<0.05). Compared with the model group, Notoginseng Radix et Rhizoma and the positive control alleviated the pathological changes in the renal tissue, such as vacuolar and fibroid changes, glomerulus atrophy, cystic expansion of renal tubules, and massive infiltration of inflammatory cells. Compared with the normal group, the model group showed decreased mitochondrial ATP content and Na~+-K~+-ATPase and Ca~(2+)-Mg~(2+)-ATPase activities in the renal tissue(P<0.05), and medium-and high-dose Notoginseng Radix et Rhizoma and positive control mitigated such decreases(P<0.05). Compared with the model group, medium-and high-dose Notoginseng Radix et Rhizoma and the positive control up-regulated the protein levels of ATP5B and SHIP2 and down-regulated the protein levels of mTORC1, P70S6K, P85, Akt, and p-Akt(P<0.05 or P<0.01 or P<0.001). Notoginseng Radix et Rhizoma may exert an anti-fibrosis effect by inhibiting the activation of the PI3K/Akt/mTORC1 pathway to restore mitochondrial energy metabolism, thus protecting the kidney.

Actin-binding protein profilin1 is an important determinant of cellular phosphoinositide control

Membrane polyphosphoinositides (PPIs) are lipid-signaling molecules that undergo metabolic turnover and influence a diverse range of cellular functions. PPIs regulate the activity and/or spatial localization of a number of actin-binding proteins (ABPs) through direct interactions; however, it is much less clear whether ABPs could also be an integral part in regulating PPI signaling. In this study, we show that ABP profilin1 (Pfn1) is an important molecular determinant of the cellular content of PI(4,5)P2 (the most abundant PPI in cells). In growth factor (EGF) stimulation setting, Pfn1 depletion does not impact PI(4,5)P2 hydrolysis but enhances plasma membrane (PM) enrichment of PPIs that are produced downstream of activated PI3-kinase, including PI(3,4,5)P3 and PI(3,4)P2, the latter consistent with increased PM recruitment of SH2-containing inositol 5' phosphatase (SHIP2) (a key enzyme for PI(3,4)P2 biosynthesis). Although Pfn1 binds to PPIs invitro, our data suggest that Pfn1's affinity to PPIs and PM presence in actual cells, if at all, is negligible, suggesting that Pfn1 is unlikely to directly compete with SHIP2 for binding to PM PPIs. Additionally, we provide evidence for Pfn1's interaction with SHIP2 in cells and modulation of this interaction upon EGF stimulation, raising an alternative possibility of Pfn1 binding as a potential restrictive mechanism for PM recruitment of SHIP2. In conclusion, our findings challenge the dogma of Pfn1's binding to PM by PPI interaction, uncover a previously unrecognized role of Pfn1 in PI(4,5)P2 homeostasis and provide a new mechanistic avenue of how an ABP could potentially impact PI3K signaling byproducts in cells through lipid phosphatase control.

Structure-Activity Studies on Bis-Sulfonamide SHIP1 Activators.

The SH2-containing inositol polyphosphate 5-phosphatase 1 (SHIP1) enzyme opposes the activity of PI3K and therefore is of interest in the treatment of inflammatory disorders. Recent results also indicate that SHIP1 promotes phagolysosomal degradation of lipids by microglia, suggesting that the enzyme may be a target for the treatment of Alzheimer's disease. Therefore, small molecules that increase SHIP1 activity may have benefits in these areas. Recently we discovered a bis-sulfonamide that increases the enzymatic activity of SHIP1. A series of similar SHIP1 activators have been synthesized and evaluated to determine structure-activity relationships and improve in vivo stability. Some new analogs have now been found with improved potency. In addition, both the thiophene and the thiomorpholine in the parent structure can be replaced by groups without a low valent sulfur atom, which provides a way to access activators that are less prone to oxidative degradation.

The Role of C2 Domains in Two Different Phosphatases: PTEN and SHIP2.

Phosphatase and tensin homologue (PTEN) and SH2-containing inositol 5'-phosphatase 2 (SHIP2) are structurally and functionally similar. They both consist of a phosphatase (Ptase) domain and an adjacent C2 domain, and both proteins dephosphorylate phosphoinositol-tri(3,4,5)phosphate, PI(3,4,5)P3; PTEN at the 3-phophate and SHIP2 at the 5-phosphate. Therefore, they play pivotal roles in the PI3K/Akt pathway. Here, we investigate the role of the C2 domain in membrane interactions of PTEN and SHIP2, using molecular dynamics simulations and free energy calculations. It is generally accepted that for PTEN, the C2 domain interacts strongly with anionic lipids and therefore significantly contributes to membrane recruitment. In contrast, for the C2 domain in SHIP2, we previously found much weaker binding affinity for anionic membranes. Our simulations confirm the membrane anchor role of the C2 domain in PTEN, as well as its necessity for the Ptase domain in gaining its productive membrane-binding conformation. In contrast, we identified that the C2 domain in SHIP2 undertakes neither of these roles, which are generally proposed for C2 domains. Our data support a model in which the main role of the C2 domain in SHIP2 is to introduce allosteric interdomain changes that enhance catalytic activity of the Ptase domain.

Effects of Nemacystus decipiens polysaccharide on mice with antibiotic associated diarrhea and colon inflammation.

Antibiotic associated diarrhea (AAD) is a common side effect of antibiotic therapy in which gut microbiota plays an important role in the disease. However, the function of gut microbiota in this disease is still not entirely clear. Polysaccharides have shown strong activity in shaping gut microbiota. Whether the polysaccharide can intervene with the microbiota to improve ADD has not been determined. In this study, we extract crude polysaccharides from Nemacystus decipiens (N. decipiens), a traditional Chinese medicine (TCM), named NDH0. The crude polysaccharide NDH0 might significantly relieve the symptom of mice with AAD, including a reduction in body weight, shortening of cecum index and the infiltration of inflammatory cells into the colon. NDH0-treated mice exhibited more abundant gut microbial diversity; significantly increased the abundance of Muribaculum, Lactobacillus, and Bifidobacterium and decreased the abundance of Enterobacter and Clostridioides at genus level. NDH0 treatment down-regulated the level of pro-inflammatory cytokines, including IL-1β and IL-6 in colon tissue. NDH0 protected the integrity of colon tissues and partially inactivated the related inflammation pathway by maintaining occludin and SH2-containing Inositol 5'-Phosphatase (SHIP). NDH0 could alleviate symptoms of diarrhea by modulating gut microbiota composition, improving intestinal integrity and reducing inflammation. The underlying protective mechanism was to reduce the abundance of opportunistic pathogens and maintain SHIP protein expression. Collectively, our results demonstrated the role of NDH0 as a potential intestinal protective agent in gut dysbiosis.

Reduced expression and activity of patient-derived SHIP1 phosphatase domain mutants

The characterization of dysregulated proteins in cell signaling pathways is important for the development of therapeutic approaches. The PI3K/AKT/mTOR pathway is frequently upregulated in cancer cells and the SH2-containing inositol 5-phosphatase SHIP1 can act as a negative regulator of the PI3K/AKT pathway. In this study, we investigated different patient-derived mutations within the conserved phosphatase domain of SHIP1. We could demonstrate that 2 out of 7 SHIP1-phosphatase domain mutations (G585K and R673Q) possessed reduced protein expression and reduced enzymatic activity in comparison to SHIP1 wild type (WT) protein and two additional mutations (E452K, R551Q) possessed reduced enzymatic activity at a comparable expression level compared to SHIP1 WT in the cell line H1299. The investigated mutations resulted in protein expression levels that were up to 93% lower than those of the SHIP1 WT for SHIP1 mutant R673Q and the enzymatic activity was below the detection limit of the performed phosphatase assay. Whereas the protein level of the R673Q mutant was reduced in comparison to SHIP1 WT the mRNA level was comparable indicating a post-transcriptional regulation. SHIP1 R673Q was rapidly degraded, with a calculated half-life of l.5 h. In addition, SHIP1 R673Q levels were significantly increased by the treatment with the proteasome inhibitor MG-132 in comparison to the DMSO control. Therefore, SHIP1 was confirmed as the target of enhanced proteasomal degradation. Computational analysis of the wild type and mutant protein structures revealed that the loss of the positively charged arginine residue R673 is associated with the loss of two salt bridges to the negatively charged amino acids D617 and E634 leading to an intramolecular instability of the mutated SHIP1 R673Q protein.Six out of seven SHIP1 mutants significantly affected the PI3K/AKT/mTOR pathway in the three cancer cell lines H1299, Reh and Sem. Four out of seven SHIP1 mutants affected phosphorylation of AKT and its target GSK3β positively compared to SHIP1 WT, whereas a negative effect on the phosphorylation of S6 was found in five out of seven mutants. In general, SHIP1 mutants impacting signal transduction were either associated with decreased SHIP1 activity or SHIP1 expression or both.Overall, the presented results indicate a regulation of the protein expression and activity of SHIP1 by patient-derived mutations in its phosphatase domain.

Abelson tyrosine kinase controls BCR signalling and B-cell differentiation by promoting B-cell metabolism.

As a nonreceptor tyrosine kinase, Abelson tyrosine kinase (c-Abl) was first studied in chronic myelogenous leukaemia, and its role in lymphocytes has been well characterised. c-Abl is involved in B-cell development and CD19-associated B-cell antigen receptor (BCR) signalling. Although c-Abl regulates different metabolic pathways, the role of c-Abl is still unknown in B-cell metabolism. In this study, B-cell-specific c-Abl knockout (KO) mice (Mb1Cre+/- c-Ablfl/fl ) were used to investigate how c-Abl regulates B-cell metabolism and BCR signalling. We found that the levels of activation positive BCR signalling proximal molecules, phosphorylated spleen tyrosine kinase (pSYK) and phosphorylated Bruton tyrosine kinase (pBTK), were decreased, while the level of key negative regulator, phosphorylated SH2-containing inositol phosphatase 1 (pSHIP1), was increased in Mb1Cre+/- c-Ablfl/fl mice. Furthermore, we found c-Abl deficiency weakened the B-cell spreading, formation of BCR signalosomes, and the polymerisation of actin during BCR activation, and also impaired the differentiation of germinal center (GC) B-cells both in quiescent condition and after immunisation. Moreover, B-cell mitochondrial respiration and the expression of B-cell metabolism-regulating molecules were downregulated in c-Abl deficiency mice. Overall, c-Abl, which involved in actin remodelling and B-cell metabolism, positively regulates BCR signalling and promotes GC differentiation.

MicroRNA networks in FLT3-ITD acute myeloid leukemia

MiR-126 and miR-155 are key microRNAs (miRNAs) that regulate, respectively, hematopoietic cell quiescence and proliferation. Herein we showed that in acute myeloid leukemia (AML), the biogenesis of these two miRNAs is interconnected through a network of regulatory loops driven by the FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD). In fact, FLT3-ITD induces the expression of miR-155 through a noncanonical mechanism of miRNA biogenesis that implicates cytoplasmic Drosha ribonuclease III (DROSHA). In turn, miR-155 down-regulates SH2-containing inositol phosphatase 1 (SHIP1), thereby increasing phosphor-protein kinase B (AKT) that in turn serine-phosphorylates, stabilizes, and activates Sprouty related EVH1 domain containing 1 (SPRED1). Activated SPRED1 inhibits the RAN/XPO5 complex and blocks the nucleus-to-cytoplasm transport of pre-miR-126, which cannot then complete the last steps of biogenesis. The net result is aberrantly low levels of mature miR-126 that allow quiescent leukemia blasts to be recruited into the cell cycle and proliferate. Thus, miR-126 down-regulation in proliferating AML blasts is downstream of FLT3-ITD–dependent miR-155 expression that initiates a complex circuit of concatenated regulatory feedback (i.e., miR-126/SPRED1, miR-155/human dead-box protein 3 [DDX3X]) and feed-forward (i.e., miR-155/SHIP1/AKT/miR-126) regulatory loops that eventually converge into an output signal for leukemic growth.

Expression, purification and characterization of the RhoA-binding domain of human SHIP2 in E.coli

Human SH2-containing inositol 5-phosphatase 2 (SHIP2) is a multi-domain protein playing essential roles in various physiological and pathological processes. In cell polarization and migration, SHIP2 serves as a RhoA effector for manipulating the level of phosphatidylinositol 3,4,5-trisphosphate. The domain between SH2 and a potential PH-R domain of SHIP2 was suggested to bind with GTP-bound form of RhoA. However, the structure of this RhoA-binding domain (RBD) of SHIP2 and the mechanism for its binding with RhoA remain unknown. In this study, SHIP2118−298 and SHIP2176−298, two truncated proteins harboring the RBD were designed, expressed, and purified successfully in E. coli. Unexpectedly, both SHIP2118−298 and SHIP2176−298 were determined to exist as homo-dimers in solution by multi-angle light scattering. Circular dichroism spectra indicated that both proteins predominantly consisted of α-helix structure. Moreover, in pull-down experiments, both proteins could bind with GTP-bound RhoA and RhoAQ63L, a mutant mimicing the state of GTP-bound RhoA. Importantly, in silico analysis showed that the shorter truncation, SHIP2176−298, contained all ordered residues between the SH2 and the PH-R domain, and matched the RhoA effector motif 1 of PKN1 well in sequence alignment, suggesting that SHIP2176−298 is sufficient for further studies on the structure and RhoA binding of SHIP2. This work shortens and confirms the main region of SHIP2 interacting with RhoA, provides the method for sample preparation, and presents preliminary information for SHIP2-RBD structure, which will facilitate the comprehensive understanding of the structure and function of SHIP2.

Overexpression of SH2-Containing Inositol Phosphatase Contributes to Chronic Lymphocytic Leukemia Survival.

Balanced activity of kinases and phosphatases downstream of the BCR is essential for B cell differentiation and function and is disturbed in chronic lymphocytic leukemia (CLL). In this study, we employed IgH.TEμ mice, which spontaneously develop CLL, and stable EMC CLL cell lines derived from these mice to explore the role of phosphatases in CLL. Genome-wide expression profiling comparing IgH.TEμ CLL cells with wild-type splenic B cells identified 96 differentially expressed phosphatase genes, including SH2-containing inositol phosphatase (Ship2). We found that B cell-specific deletion of Ship2, but not of its close homolog Ship1, significantly reduced CLL formation in IgH.TEμ mice. Treatment of EMC cell lines with Ship1/2 small molecule inhibitors resulted in the induction of caspase-dependent apoptosis. Using flow cytometry and Western blot analysis, we observed that blocking Ship1/2 abrogated EMC cell survival by exerting dual effects on the BCR signaling cascade. On one hand, specific Ship1 inhibition enhanced calcium signaling and thereby abrogated an anergic response to BCR stimulation in CLL cells. On the other hand, concomitant Ship1/Ship2 inhibition or specific Ship2 inhibition reduced constitutive activation of the mTORC1/ribosomal protein S6 pathway and downregulated constitutive expression of the antiapoptotic protein Mcl-1, in both EMC cell lines and primary IgH.TEμ CLL cells. Importantly, also in human CLL, we found overexpression of many phosphatases including SHIP2. Inhibition of SHIP1/SHIP2 reduced cellular survival and S6 phosphorylation and enhanced basal calcium levels in human CLL cells. Taken together, we provide evidence that SHIP2 contributes to CLL pathogenesis in mouse and human CLL.

Monomer hapten and hapten-specific IgG inhibit mast cell activation evoked by multivalent hapten with different mechanisms.

Aggregation of IgE bound to high affinity IgE receptor (FcεRI) by multivalent antigen induces mast cell activation. Reportedly, disaggregation of aggregated FcεRI immediately terminated degranulation, and formation of co-ligated FcεRI and low affinity IgG receptor FcγRIIB blocked degranulation by inhibitory signal via SH2-containing inositol 5'-phosphatase 1 (SHIP1) phosphorylation. However, their molecular mechanisms to inhibit mast cell activation have been unclear in detail. Herein, we found that addition of excess monomeric hapten (TNP-alanine) to multivalent antigen (TNP-OVA)-activated rat basophilic leukemia cells and mouse bone marrow-derived mast cells induced immediate and transient Syk dephosphorylation, which was previously phosphorylated by TNP-OVA addition. Syk dephosphorylation correlated to rapidly decreased intracellular Ca2+ concentrations ([Ca2+ ]i ), terminated degranulation, and suppressed cytokine production through inhibition of Akt and ERK phosphorylation. Addition of hapten-specific IgG monoclonal antibody (anti-TNP IgG1) to activated mast cells induced translocation of SHIP1 to the plasma membrane and its phosphorylation, indicating that co-ligation of FcεRI and FcγRIIB after FcεRI aggregation can lead to SHIP1 activation. SHIP1 phosphorylation led to gradually decreased [Ca2+ ]i , weak inhibition of degranulation, and strong inhibition of cytokine production. Our findings clearly show the inhibitory mechanism of cell function in activated mast cells by operating Fc receptor crosslinking.

Dedicator of cytokinesis protein 2 couples with lymphoid enhancer–binding factor 1 to regulate expression of CD21 and B-cell differentiation

B-cell receptor (BCR) signaling, combined with CD19 and CD21 signals, imparts specific control of B-cell responses. Dedicator of cytokinesis protein 2 (DOCK2) is critical for the migration and motility of lymphocytes. Although absence of DOCK2 leads to lymphopenia, little is known about the signaling mechanisms and physiologic functions of DOCK2 in B cells. We sought to determine the underlying molecular mechanism of how DOCK2 regulates BCR signaling and peripheral B-cell differentiation. In this study we used genetic models for DOCK2, Wiskott-Aldrich syndrome protein (WASP), and lymphoid enhancer-binding factor 1 deficiency to study their interplay in BCR signaling and B-cell differentiation. We found that the absence of DOCK2 led to downregulation of proximal and distal BCR signaling molecules, including CD19, but upregulation of SH2-containing inositol 5 phosphatase 1, a negative signaling molecule. Interestingly, DOCK2 deficiency reduced CD19 and CD21 expression at the mRNA and/or protein levels and was associated with reduced numbers of marginal zone B cells. Additionally, loss of DOCK2 reduced activation of WASP and accelerated degradation of WASP, resulting into reduced actin accumulation and early activation of B cells. Mechanistically, the absence of DOCK2 upregulates the expression of lymphoid enhancer-binding factor 1. These differences were associated with altered humoral responses in the absence of DOCK2. Overall, our study has provided a novel underlying molecular mechanism of how DOCK2 deficiency regulates surface expression of CD21, which leads to downregulation of CD19-mediated BCR signaling and marginal zone B-cell differentiation.

Analysis of the FLVR motif of SHIP1 and its importance for the protein stability of SH2 containing signaling proteins

Binding of proteins with SH2 domains to tyrosine-phosphorylated signaling proteins is a key mechanism for transmission of biological signals within the cell. Characterization of dysregulated proteins in cell signaling pathways is important for the development of therapeutic approaches. The AKT pathway is a frequently upregulated pathway in most cancer cells and the SH2-containing inositol 5-phosphatase SHIP1 is a negative regulator of the AKT pathway. In this study we investigated different mutations of the conserved FLVR motif of the SH2 domain and putative phosphorylation sites of SHIP1 which are located in close proximity to its FLVR motif. We demonstrate that patient-derived SHIP1-FLVR motif mutations e.g. F28L, and L29F possess reduced protein expression and increased phospho-AKT-S473 levels in comparison to SHIP1 wildtype. The estimated half-life of SHIP1-F28L protein was reduced from 23.2 h to 0.89 h in TF-1 cells and from 4.7 h to 0.6 h in Jurkat cells. These data indicate that the phenylalanine residue at position 28 of SHIP1 is important for its stability. Replacement of F28 with other aromatic residues like tyrosine and tryptophan preserves protein stability while replacement with non-aromatic amino acids like leucine, isoleucine, valine or alanine severely affects the stability of SHIP1. In consequence, a SHIP1-mutant with an aromatic amino acid at position 28 i.e. F28W can rescue the inhibitory function of wild type SHIP1, whereas SHIP1-mutants with non-aromatic amino acids i.e. F28V do not inhibit cell growth anymore. A detailed structural analysis revealed that F28 forms hydrophobic surface contacts in particular with W5, I83, L97 and P100 which can be maintained by tyrosine and tryptophan residues, but not by non-aromatic residues at position 28. In line with this model of mutation-induced instability of SHIP1-F28L, treatment of cells with proteasomal inhibitor MG132 was able to rescue expression of SHIP1-F28L. In addition, mutation of putative phosphorylation sites S27 and S33 adjacent to the FLVR motif of SHIP1 have an influence on its protein stability. These results further support a functional role of SHIP1 as tumor suppressor protein and indicate a regulation of protein expression of SH2 domain containing proteins via the FLVR motif.

Lipid phosphatase SHIP-1 regulates chondrocyte hypertrophy and skeletal development.

SH2‐containing inositol‐5′‐phosphatase‐1 (SHIP‐1) controls the phosphatidylinositol‐3′‐kinase (PI3K) initiated signaling pathway by limiting cell membrane recruitment and activation of Akt. Despite the fact that many of the growth factors important to cartilage development and functions are able to activate the PI3K signal transduction pathway, little is known about the role of PI3K signaling in chondrocyte biology and its contribution to mammalian skeletogenesis. Here, we report that the lipid phosphatase SHIP‐1 regulates chondrocyte hypertrophy and skeletal development through its expression in osteochondroprogenitor cells. Global SHIP‐1 knockout led to accelerated chondrocyte hypertrophy and premature formation of the secondary ossification center in the bones of postnatal mice. Drastically higher vascularization and greater number of c‐kit + progenitors associated with sinusoids in the bone marrow also indicated more advanced chondrocyte hypertrophic differentiation in SHIP‐1 knockout mice than in wild‐type mice. In corroboration with the in vivo phenotype, SHIP‐1 deficient PDGFRα + Sca‐1 + osteochondroprogenitor cells exhibited rapid differentiation into hypertrophic chondrocytes under chondrogenic culture conditions in vitro. Furthermore, SHIP‐1 deficiency inhibited hypoxia‐induced cellular activation of Akt and extracellular‐signal‐regulated kinase (Erk) and suppressed hypoxia‐induced cell proliferation. These results suggest that SHIP‐1 is required for hypoxia‐induced growth signaling under physiological hypoxia in the bone marrow. In conclusion, the lipid phosphatase SHIP‐1 regulates skeletal development by modulating chondrogenesis and the hypoxia response of the osteochondroprogenitors during endochondral bone formation.

Inhibiting phosphatase SHIP-1 enhances suboptimal IgE-mediated activation of human blood basophils but inhibits IgE-mediated activation of cultured human mast cells

IgE-mediated activation of basophil granulocytes and mast cells follows a bell-shaped dose-response curve. The decreased activation at supraoptimal allergen stimulation is thought to be associated with SH2-containing inositol-5′-phosphatase 1 (SHIP-1). SHIP-1 phosphorylation is inversely related to IgE-mediated releasability of basophils. This study sought to clarify the regulatory role of SHIP-1 in degranulation of basophil granulocytes and mast cells by selective inhibition of the phosphatase function of SHIP-1with 3-α-aminocholestane (3-α-AC).Six grass pollen allergic patients, six non-responder patients and six cultured human primary mast cell lines were included. The effect of 3-α-AC (1–60 μM, 30 min, 37 °C) was analyzed at individual suboptimal, optimal and supra-optimal allergen concentrations. The activity, upregulation of CD63, measured at different conditions was compared to evaluate the maximal effect of selective SHIP-1 inhibition. Basophils of five non-responder patients were treated with 3-α-AC (10 μM, 30 min, 37 °C).At high concentrations (>60 μM) of 3-α-AC, cells appeared to enter apoptosis. The median reactivity increased from 27.1% to 44.9% CD63+ basophils at 10 μM of 3-α-AC and suboptimal allergen stimulation (p = 0.0153). There was no effect on blood basophils of 3-α-AC at optimal or supra-optimal allergen concentrations. In contrast, treatment with more than 6 μM 3-α-AC significantly inhibited mast cell reactivity. 10 μM 3-α-AC reduced median reactivity from 32.85% to 16.5% CD63+ mast cells (p = 0.0465). Treatment with 3-α-AC did not increase response of basophils of non-responder patients.Modulating blood basophils with 3-α-AC enhanced reactivity only at suboptimal allergen concentration, and basophils from non-responders did not regain responsiveness to IgE stimulation. 3-α-AC inhibited the IgE response of mast cells in a dose dependent manner.

Stimulus strength determines the BTK-dependence of the SHIP1-deficient phenotype in IgE/antigen-triggered mast cells

Antigen (Ag)-mediated crosslinking of IgE-loaded high-affinity receptors for IgE (FcεRI) on mast cells (MCs) triggers activation of proinflammatory effector functions relevant for IgE-associated allergic disorders. The cytosolic tyrosine kinase BTK and the SH2-containing inositol-5′-phosphatase SHIP1 are central positive and negative regulators of Ag-triggered MC activation, respectively, contrarily controlling Ca2+ mobilisation, degranulation, and cytokine production. Using genetic and pharmacological techniques, we examined whether BTK activation in Ship1−/− MCs is mandatory for the manifestation of the well-known hyperactive phenotype of Ship1−/− MCs. We demonstrate the prominence of BTK for the Ship1−/− phenotype in a manner strictly dependent on the strength of the initial Ag stimulus; particular importance for BTK was identified in Ship1−/− bone marrow-derived MCs in response to stimulation with suboptimal Ag concentrations. With respect to MAPK activation, BTK showed particular importance at suboptimal Ag concentrations, allowing for an analogous-to-digital switch resulting in full activation of ERK1/2 already at low Ag concentrations. Our data allow for a more precise definition of the role of BTK in FcεRI-mediated signal transduction and effector function in MCs. Moreover, they suggest that reduced activation or curtate expression of SHIP1 can be compensated by pharmacological inhibition of BTK and vice versa.

IgG-Independent Co-aggregation of FcεRI and FcγRIIB Results in LYN- and SHIP1-Dependent Tyrosine Phosphorylation of FcγRIIB in Murine Bone Marrow-Derived Mast Cells.

Activation of the high-affinity receptor for IgE (FcεRI) follows a bell-shaped dose-response curve. Upon supra-optimal stimulation, mast cell effector responses are down-regulated by inhibitory molecules like the SH2-containing inositol-5′-phosphatase SHIP1 and the SRC-family-kinase LYN. To identify further molecules involved in a negative regulatory signalosome, we screened for proteins showing the same pattern of tyrosine phosphorylation as SHIP1, which is tyrosine-phosphorylated strongest upon supra-optimal antigen (Ag) stimulation. The low-affinity IgG receptor, FcγRIIB, was found to be most strongly phosphorylated under supra-optimal conditions. This phosphorylation is the consequence of passive, Ag/IgE-dependent and progressive co-localization of FcεRI and FcγRIIB, which is not dependent on IgG. Upon supra-optimal FcεRI cross-linking, FcγRIIB phosphorylation is executed by LYN and protected from dephosphorylation by SHIP1. Analysis of FcγRIIB-deficient bone marrow-derived mast cells revealed an ambiguous phenotype upon FcεRI cross-linking. Absence of FcγRIIB significantly diminished the level of SHIP1 phosphorylation and resulted in augmented Ca2+ mobilization. Though, degranulation and IL-6 production were only weakly altered. Altogether our data establish the LYN/FcγRIIB/SHIP1 signalosome in the context of FcεRI activation, particularly at supra-optimal Ag concentrations. The fact that SHIP1 tyrosine phosphorylation/activation not only depends on FcγRIIB, highlights the necessity for its tight backup control.