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Regulation of proliferation, survival, differentiation, and activation by the Signaling Platform for SHP-1 phosphatase
Advances in Enzyme Regulation 52 (2012) 7–15
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Advances in Enzyme Regulation journal homepage: www.elsevier.com/locate/ advenzreg
Regulation of proliferation, survival, differentiation, and activation by the Signaling Platform for SHP-1 phosphatase Toshiaki Kawakami*, Wenbin Xiao 1, Hiroki Yasudo, Yuko Kawakami Division of Cell Biology, La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037, USA
Introduction The recognition of a substrate by an enzyme has been taught as being very specific like ‘lock and key’ in a textbook of biochemistry. However, we now know that an enzyme can recognize various substrates. For example, in cell-free reactions, many protein-tyrosine kinases can phosphorylate different substrates ranging from an authentic in vivo substrate to artificial peptides such as poly(Glu– Tyr). Protein phosphatases can also dephosphorylate non-specific substrates such as p-nitrophenyl phosphate. The loose specificity in enzyme-substrate recognition would potentially create serious problems in many physiological processes such as metabolism, transcription, cell cycle progression, survival/apoptosis, differentiation, cytoskeletal rearrangement/cell movement, etc. However, mechanisms to enhance the specificity in enzyme-substrate recognition have evolved in the signal transduction pathways underpinning these functions. Some enzymes might have extremely high specificity, similar to that of antibody recognition of antigen. However, this strategy that simply increases the specificity might not be sufficient to ensure the efficient, fail-safe flow of information in signal transduction within a cell. Another strategy developed in evolution is restricting the enzyme-substrate interaction using an adaptor molecule. The prototypical example of this mechanism is found in the mitogen-activated protein kinase (MAPK) cascade: MAPK modules are composed of a cascade of three intracellular protein kinases (MKKK, MKK and MAPK) which are activated successively by phosphorylation events (Brunet and Pouyssegur, 1997; Burack and Shaw, 2000). In addition to the molecular selectivity of each enzyme for its substrate, exogenous proteins, such as the yeast Ste5 protein, serve as ‘chaperone’ proteins to tether all the members of a module and restrict signal transduction to this module. In addition to the efficient and accurate information flow, the adaptor-aided signaling cassette can direct the location and timing of enzymatic reactions, two factors essential for signal transduction. The human genome contains 518 genes encoding protein kinases (Manning et al., 2002) and 107 genes encoding protein-tyrosine phosphatases (Alonso et al., 2004). The SH2 domain-containing
* Corresponding author. E-mail address: [email protected] (T. Kawakami). 1 Present address: Department of Pathology, University Hospitals Case Medical Center, Case Western Reserve University, Cleveland, OH 44106, USA. 0065-2571/$ – see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.advenzreg.2011.09.003
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protein phosphatase 1 (SHP-1) is a protein-tyrosine phosphatase expressed in hematopoietic cells. SHP-1 has two tandem SH2 domains at the N-terminus, followed by a catalytic domain, and an inhibitory C-terminus. SHP-1 has a negative impact on lymphocyte signaling (Tsui et al., 2006). By contrast, its cousin SHP-2, which is widely expressed, in general promotes signaling pathways that lead to differentiation, cell growth, and migration (Lorenz, 2009; Poole and Jones, 2005; Tartaglia et al., 2004). In this review, we will summarize recent studies on how SHP-1 activity is regulated through formation of multimolecular complexes. Phospholipase C-b3 is a novel tumor suppressor Phospholipase C (PLC)-b is a small family of four enzymes that can produce diacylglycerol and inositol 1,4,5-trisphosphate (IP3) downstream of heterotrimeric G proteins (Rhee, 2001). As diacylglycerol can activate several isoforms of protein kinase C (PKC) (Nishizuka, 1992), and IP3 can mobilize Ca2þ (Berridge, 1993), PLC-b is implicated in promoting cell proliferation. PLC-b directly interacts with GTP-bound Ga subunits, leading to its catalytic activation. A recent study identified three Gaq-binding regions in PLC-b3 (Waldo et al., 2010): the short helix-turn-helix (Ha1 and Ha2) region following the C2 domain and the loop between the catalytic TIM barrel and C2 domain are critical for PLC catalytic activity; the third Gaq-interacting region, EF hands 3 and 4, are critical for GTP hydrolysis by Gaq. PLC-b2 and PLC-b3 can also be activated by bg subunits of the Gai/o family of G proteins (Camps et al., 1992; Katz et al., 1992; Lee et al., 1993). Consistent with their roles in G protein-coupled receptor signaling, chemokine-induced IP3 production, Ca2þ signaling, and migration are reduced in PLC-b2/ and PLC-b2/; PLC-b3/ neutrophils (Li et al., 2000) and T cells (Bach et al., 2007). The role of PLC-b3 in tumorigenesis has not been noticed until recently. PLC-b3 deficient mice die prematurely, mostly (w90%) by developing myeloproliferative neoplasm (MPN), which evolves to accelerated and blast-crisis stages. Some mice develop other tumors such as T cell lymphoma, carcinomas of skin and lung, and hamartoma (Xiao et al., 2009). This phenomenon is unique to PLC-b3/ mice, as PLCb2/ mice do not develop tumors or die prematurely. Aged PLC-b3/ mice with splenomegaly typically have increased numbers of c-KitþSca-1þLineage cells (KSL cells; enriched for hematopoietic stem cells [HSCs]), granulocyte-macrophage progenitors, and megakaryocyte-erythroid progenitors in the bone marrow (BM) and spleens. Consistent with these immunophenotype data, PLC-b3/ BM cells and splenocytes give rise to greater numbers of myeloid colonies than WT cells in methylcellulose medium, suggesting that PLC-b3/ HSCs and myeloid progenitors have an increased predisposition to differentiate into granulocytes. Moreover, PLC-b3/ BM and KSL cells are hypersensitive to cytokines such as GM-CSF and IL-3, a hallmark of human MPNs (Emanuel et al., 1991), and form macrophage and granulocytemacrophage colonies in the absence of growth factors, a feature characteristic of transformed cells. In vivo BrdU incorporation, in vitro cultures, and cell cycle analysis of KSL cells demonstrate increased proliferation in PLC-b3/ HSC-enriched populations (Xiao et al., 2009). Similarly, siRNAmediated silencing of PLC-b3 leads to enhanced proliferation in vascular endothelial growth factorstimulated endothelial cells (Bhattacharya et al., 2009). Apoptosis is less abundant in PLC-b3/ KSL cells. The MPN is transplantable with long-term HSC-enriched CD34 KSL cells derived from PLC-b3/ mice, indicating that the leukemic stem cells responsible for the development of MPD in PLC-b3/ mice are present in CD34 KSL cells. An interesting question is whether the microenviroment plays any role in the development of MPN in PLC-b3/ mice. The long latency of MPN in these mice probably is related to the microenviroment. However, when wild-type (wt) BM or HSCs were transferred into irradiated PLC-b3/ mice, no MPN was observed, suggesting that the microeviroment in PLC-b3/ mice is not sufficient for MPN development. Flow cytometry and immunofluorescence microscopy show that Stat5-Tyr694 phosphorylation is constitutively increased and localized in the nuclei of PLC-b3/ KSL cells. By contrast, Stat3 phosphorylation is comparable in PLC-b3/ and WT cells. Lymphomas and skin carcinoma from PLC-b3/ mice also exhibit increased phosphorylation of Stat5. Importantly, introduction of dominant-negative (DN) Stat5 into PLC-b3/ KSL cells suppresses their in vitro expansion, colony-forming ability, and MPN induction in transplanted mice. These results demonstrate that the increased Stat5 activity is responsible for the development of MPN (and probably other tumors as well) in PLC-b3/ mice. Thus, PLC-b3 is a novel tumor suppressor (Xiao et al., 2009).
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SPS complex The findings that PLC-b3 can suppress Stat5 activity were unexpected, as PLC-b3 is conventionally thought to be a component of GPCR signaling pathway. Structure-function analysis showed that the Cterminal portion of PLC-b3 (PLC-b3-CT, positions 809–1234) has the growth-inhibitory activity in IL-3dependent mouse Ba/F3 cells. Remarkably, we discovered a novel multimolecular complex termed SPS complex, which is composed of SHP-1, PLC-b3, and Stat5 (a transcription factor essential for proliferation and survival of myeloid cells) (Xiao et al., 2009). SHP-1 and Stat5 interact with PLC-b3 via the PLCb3-CT. The physical proximity of SHP-1 (enzyme) and Stat5 (substrate) brought about by these interactions allows an efficient dephosphorylation of Stat5 at Tyr496, the critical site of phosphorylation for Stat5 activation (Gouilleux et al., 1994). Since more than 80% of the three molecules can be found in the SPS complex after 5 min stimulation of Ba/F3 cells with IL-3, the Stat5-Tyr496 dephosphorylation is considered a major mechanism to keep Stat5 activation in check. Deficiency of PLC-b3 in knockout mice (Xiao et al., 2009) or loss-of-function mutation of SHP-1 in viable motheaten mice (Tsui et al., 1993) loses this mechanism to allow constitutive activation of Stat5 and MPN development. Interestingly, SHP-1 does not appear to dephosphorylate STAT3 in some types of leukemia cells (McKenzie et al., 2011), which may explain unchanged phosphorylation status of Stat3 in PLC-b3/ cells. PLC-b3 haploinsufficiency cooperates with c-myc for malignant transformation Haploinsufficiency is a typical feature of many classic tumor suppressors. Since we found that c-myc can induce in vitro transformation in PLC-b3þ/ and PLC-3/ mouse embryonic fibroblasts, we investigated whether PLC-b3 deficiency can cooperate with c-myc to induce in vivo tumor formation. Compared to Em-myc transgenic mice that develop B cell-lineage lymphomas with a long latency (Adams et al., 1985), pre-B cell lymphoma formation in Em-myc;PLC-b3þ/ mice was dramatically accelerated. These Em-myc;PLC-3þ/ lymphomas and PLC-b3/ lymphomas showed high levels of Stat5 phosphorylation. Importantly, all analyzed PLC-b3/ lymphomas showed as high c-myc expression as did Em-myc;PLC-b3þ/ lymphomas. Retroviral expression of DN Stat5 or PLC-b3-CT in Em-myc;PLC-3þ/ lymphoma cells suppressed their in vitro growth and colony formation, indicating that PLC-b3 haploinsufficiency cooperates with c-myc to transform fibroblasts and lymphocytes. Translocations of c-myc to immunoglobulin or other gene loci and thus abnormal expression of c-myc are causally linked to Burkitt’s lymphoma (Boxer and Dang, 2001). Interestingly, two of six Burkitt’s lymphoma cell lines tested, i.e., Daudi and Raji, exhibited very low levels of PLC-b3 protein and high levels of STAT5 phosphorylation. Overexpression of PLC-b3-CT or DN Stat5 in these cells blocked their growth and reduced STAT5 phosphorylation. These results suggest that reduced or abrogated expression of PLC-b3 can cooperate with active c-myc to induce lymphoma in mice and humans. Interestingly, approximately 10% percent of chronic lymphocytic leukemia samples showed low levels of PLC-b3 expression with high phospho-STAT5 levels. The results collectively suggest that reduced expression of PLC-b3 and thus the loss of the SHP-1-mediated Stat5 dephosphorylation mechanism cooperates with active c-myc to induce hematopoietic malignancies in mice and humans. LynL/L;PLC-b3L/L mice develop severe myelodysplastic syndrome (MDS)/MPN Searching for further molecular mechanisms for the increased Stat5 activity, we found dramatically increased phosphorylation of Lyn (a Src family kinase prominently expressed in myeloid and B cells) at Tyr396 in the activation loop in PLC-b3/ BM cells. Surprisingly, doubly deficient Lyn/;PLC-b3/ mice all died within 10 months after birth, with severe macrophage infiltration in the lungs (Xiao et al., 2010), similar to Lyn/;hck/ (Xiao et al., 2008), SHIP/ (Nakamura et al., 2004; Rauh et al., 2005; Xiao et al., 2008) and mev/mev (Xiao et al., 2009) mice. Myeloid cell infiltration was also seen in liver and kidney of Lyn/;PLC-b3/ mice. Comprehensive phenotype analysis indicated that unlike PLCb3/ and Lyn/ mice, Lyn/;PLC-b3/ mice had MDS/MPN with monocytosis, similar to human chronic myelomonocytic leukemia (Emanuel, 2008). Transplantation experiments showed that mice that received BM cells derived from Lyn/;PLC-3/ mice developed severe MDS/MPN with increased
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monocytes, anemia and thrombocytopenia, recapitulating the phenotype of Lyn/;PLC-b3/ mice. These results demonstrate that the MDS/MPN in Lyn/;PLC-b3/ mice is largely BM cell-autonomous. Lyn/;PLC-b3/ mice had higher levels of HSCs than PLC-b3/ and Lyn/ mice, as expected from the more severe form of MPN/MDS in the former mice. Consistent with this phenotype, Lyn/ ;PLC-b3/ KSL cells grew 2–3-fold faster than PLC-b3/ and Lyn/ cells. Cell cycle analysis revealed more Lyn/ KSL cells in the S/G2/M phases but comparable numbers in the G0 phase compared to wt cells, indicating that Lyn is a negative regulator for the G1-S transition, but has no effects on the G0-G1 checkpoint. On the other hand, compared to wt cells, both PLC-b3/ and Lyn/ ;PLC-b3/ KSL cells cycled more frequently into G1 and S/G2/M phases and stayed less frequently in the G0 phase. These results suggest that PLC-b3 is essential for regulation of the G0–G1 transition. Under low concentrations of IL-3 or GM-CSF, wt KSL cells gave rise to small numbers of colonies, and colony-forming efficiencies were significantly higher in Lyn/ and PLC-b3/ KSL cells. As expected, Lyn/;PLC-b3/ KSL cells generated the highest number of colonies, in line with their highest Stat5 activity among the four genotypes. Similar to PLC-b3/ cells, Stat5-Tyr694 phosphorylation was increased in Lyn/;PLC-3/ KSL cells before and after cytokine stimulation. Transduction with DN Stat5 showed that Stat5 is essential for the survival of these cells and that Stat5 is required for MDS/ MPN development in Lyn/;PLC-b3/ mice. Both PLC-b3 and Lyn are involved in the regulation of SHP-1 phosphorylation at Tyr536 and Tyr564 The molecular basis for the regulation of SHP-1 activity has been incompletely understood (Poole and Jones, 2005; Tsui et al., 1993). The C-terminal phosphorylation sites have been mapped to Tyr536 and Tyr564 residues (Uchida et al., 1994) and putative tyrosine kinases that catalyze these phosphorylations include Lyn (Hibbs et al., 2002; Xiao et al., 2005), Lck (Lorenz et al., 1994), and Src (Frank et al., 2004). Using phospho-specific antibodies against Tyr536 and Tyr564, we found that Tyr536 phosphorylation is reduced in both Lyn/ and PLC-b3/ cells, and is abolished in Lyn/;PLC-b3/ cells (Xiao et al., 2010). On the other hand, Tyr564 phosphorylation was not affected in PLC-b3/ cells, but reduced or almost abrogated in Lyn/ and Lyn/;PLC-b3/ cells. In vitro kinase assays confirmed that Lyn can phosphorylate SHP-1 at Tyr564 and Tyr536. These data demonstrate that Lyn is the predominant kinase that phosphorylates Tyr564, and PLC-b3 may help tyrosine kinases including Lyn to phosphorylate Tyr536 (Fig. 1). PLC-b3-CT interacts not only with SHP-1 (Xiao et al., 2009), but also with Lyn (Xiao et al., 2010) in Ba/F3 cells, splenocytes, and mast cells. Similar to PLC-b3/SHP-1 interactions, PLC-b3/Lyn interactions were enhanced by IL-3 stimulation. Furthermore, co-immunoprecipitation of Lyn with SHP-1 was also observed in wt splenocytes and PLC-b3 CT-overexpressing PLC-b3/ mast cells. Lyn/SHP-1 interactions were also enhanced by IL-3 stimulation. Interestingly, Lyn/SHP-1 interactions were only weakly detected in PLC-b3/ splenocytes and BMMCs, suggesting that PLC-b3 CT facilitates the interaction between Lyn and SHP-1. Thus, these results added a new member, i.e., Lyn, to the SPS complex (Xiao et al., 2010). Roles of SHP-1 Tyr564 and Tyr536 phosphorylation To investigate the effect of Tyr536 and Tyr564 phosphorylation on SHP-1 phosphatase activity, wt, Y536F, Y564F or doubly YF mutant (DYF) SHP-1 vectors were introduced into mev/mev CD34KSL cells. As expected, wt SHP-1 increased a phosphatase activity by 4-fold over empty vector control. Y536F mutant exhibited a phosphatase activity similar to wt enzyme. By contrast, Y564F and DYF mutants had activities similar to vector control, suggesting that Tyr564 phosphorylation is critical for SHP-1 phosphatase activity. Moreover, we found that SHP-1 phosphatase activity is inversely correlated with Stat5 activity, in vitro cell growth, and MPN-causing abilities of HSCs. Despite the normal catalytic activity, Y536F SHP-1 exhibited lower suppression of Stat5 phosphorylation and in vitro cell growth in mev/mev HSCs and mast cells, compared to wt SHP-1. This could be due to weaker interactions between Y536F SHP-1 and Stat5. Indeed, co-immunoprecipitation of SHP-1 with Stat5 was drastically reduced in mev/mev mast cells expressing Y536F or DYF SHP-1. By
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A SH2
SH2
N
PTP
Y536
N
SH2
Y564
SH2
pY536
PTP
pY564
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N Active
N
SH2
PTP
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5 at St
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P
5 at St
5 at St
Jak 2
P
Lyn
Lyn P Y564
SHP-1
PLC- β3
SHP-1
PLC- β3
PLC- β3
PLC- β3
Lyn
B
P
Y694
Fig. 1. A model for SHP-1 activation and recognition of its substrate Stat5. (A) SHP-1 is phosphorylated at Tyr564 predominantly by Lyn and at Tyr536 by Lyn and other kinase(s) including Jak2. Inactive SHP-1 (Top left) has a closed conformation with the catalytic site of the PTP domain capped by the N-SH2 domain (Yang et al., 2003). The Tyr536 phosphorylation site of SHP-1 interacts with Stat5 most probably via the SH2 domain of the latter molecule (Bottom). The Tyr564 phosphorylation site of SHP-1 interacts with N-SH2 to render the enzyme active. N-terminal ends of SHP-1 and Stat5 proteins are shown by ‘N’. (B) SHP-1 and Lyn interact with PLC-b3-CT (Xiao et al., 2010; Xiao et al., 2009). Tyr694 phosphorylated Stat5 is recruited to the PLC-b3-nucleated SPS complex containing Lyn and SHP-1, and dephosphorylated by an active SHP-1 enzyme that is phosphorylated at Tyr536 and Tyr564. Stat5 dimer is depicted as a monomer for simplicity.
contrast, wt and Y564F SHP-1 were well co-immunoprecipitated with Stat5. These results suggest that SHP-1 phosphorylation of Tyr536 is critical for interaction with Stat5. Consistent with our observation that Lyn phosphorylates SHP-1 at Tyr564, SHP-1 phosphatase activity was reduced by 40% in Lyn/ mast cells. Lyn/;PLC-b3/ cells had a very low level of SHP-1 phosphatase activity, similar to mev/mev cells (which had 10–20% activity of wt enzyme), showing that the reduced SHP-1 phosphatase activity is a common signaling defect shared by mev/mev and Lyn/;PLCb3/ mice. Although overexpression of wt SHP-1 suppressed the in vitro growth of mev/mev CD34KSL cells as well as wt, Lyn/, and PLC-b3/ cells, it had little effect on Lyn/;PLC-b3/ cells. Wt SHP-1overexpressing CD34KSL cells from Lyn/;PLC-b3/ mice still caused MPN upon transplantation. Consistent with these results, the transduced wt SHP-1 was not phosphorylated at Tyr536 or Tyr564 in Lyn/;PLC-b3/ cells. However, transduction of Lyn/;PLC-b3/ cells with SHP-1 with phosphomimetic Y536E/Y564E (DYE) mutations increased SHP-1 phosphatase activity by 10-fold in Lyn/;PLCb3/ cells, in contrast to a mere 2-fold increase by wt SHP-1 expression. Importantly, DYE-transduced cells exhibited substantially lower levels of Stat5 phosphorylation and in vitro growth rate and failed to exhibit MPN-causing properties. These results suggest that severely reduced SHP-1 phosphatase activity is responsible for the MDS/MPN-associated phenotypes observed in Lyn/;PLC-b3/ mice. Growth-inhibitory activity of short Stat5-binding peptides within PLC-b3-CT More recently, we finely mapped the Stat5- and SHP-1-binding sites within PLC-b3-CT, using affinity pulldown and a large panel of GST fusion proteins containing various portions of PLC-b3-CT
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(Yasudo et al., PLoS ONE, 2011). By designing the fusion constructs to narrow down the binding sites within less than 40 amino acid residues, we found that three noncontiguous regions, designated a (positions 917–943), b (positions 983–1000), and c (positions 1032–1069), bound to Stat5 and two regions, designated c (positions 1032–1069) and d (positions 1182–1209) bound to SHP-1. Given the critical importance of Stat5 for the proliferation, survival, and differentiation of HSCs in PLC-b3/ mice (Xiao et al., 2009), we tested whether overexpression of the Stat5-interacting, N-terminally myc-tagged peptides affects the in vitro growth of Ba/F3 cells. The experiments indicated that the Stat5-binding activity of peptides b and c may be critical for growth suppression. Because peptide b exhibited approximately 80% potency of PLC-b3-CT in Ba/F3 cell growth inhibition, we tested even shorter peptides in this region. Among them, an 11-residue peptide (peptide b11; positions 988– 998) exhibited a growth-inhibitory activity as strong as >80% that of PLC-b3-CT. As expected, Stat5 phosphorylation at Tyr694 was significantly reduced in peptide b11-expressing cells. The relevance of these in vitro studies was verified by reduced myeloid colony-forming activity of the peptide b11expressing cells and by the failure of peptide b11-expressing lin BM cells derived from PLC-b3/ mice to cause MPN development upon transplantation. PLC-b3 regulates FceRI-mediated mast cell activation Is PLC-b3-mediated regulation of SHP-1 activity relevant to other biological events? We studied this point in mast cell activation (Xiao et al., 2011). Mast cells are major effectors in high-affinity IgE receptor (FcεRI)-dependent allergic reactions (Kawakami and Galli, 2002). Activated mast cells secrete preformed proinflammatory mediators (e.g., histamine, proteases, proteoglycans, and nucleotides) as well as de novo synthesized lipids (e.g., leukotrienes and prostaglandins) and polypeptides (e.g., cytokines and chemokines). These substances lead to the development of allergic inflammation. Antigen challenge in IgE-sensitized mice induces life-threatening allergic reactions termed anaphylaxis through FcεRI-mediated mast cell activation (Inagaki et al., 1986; Wershil et al., 1991). PLC-b3/ mice showed blunted late-phase, but not acute-phase, anaphylactic responses. Consistent with this, FcεRI stimulation of PLC-b3/ mast cells exhibited substantially reduced cytokine production, but normal degranulation. Reduced cytokine production in PLC-b3/ cells could be accounted for by increased activity of the negative regulator Lyn and reduced activities of the positive regulators MAPKs. Mechanistically, PLC-b3 constitutively interacts with FcεRI, Lyn, and SHP-1. SHP-1 likely recognizes its substrates Lyn and MAPKs via the recently described kinase tyrosine-based inhibitory motif, KTIM (Abu-Dayyeh et al., 2008). Consistent with PLC-b3/SHP-1-mediated repression of Lyn kinase activity by dephosphorylation at Tyr396, FcεRI-mediated phenotypes were similar in PLC-b3/ and SHP-1-mutant mev/mev mast cells. In PLC-b3/ mast cells, Lyn is constitutively active and negatively regulates FcεRImediated activation of the lipid phosphatase SHIP-1 (Hernandez-Hansen et al., 2004; Xiao et al., 2005); phosphorylated SHIP-1 recruits Dok adaptors to activate RasGAP (Abramson et al., 2003); RasGAP negatively regulates the Ras/Raf/MEK/ERKs pathway (Mashima et al., 2009). Thus, this study defined the PLC-b3/SHP-1-mediated novel signaling pathway (Fig. 2) for FcεRI-mediated cytokine production (Xiao et al., 2011). Future perspectives Information flow between SHP-1 and Lyn is bidirectional, since SHP-1 dephosphorylates Lyn-Tyr396 and Lyn phosphorylates SHP-1 at two residues, Tyr536 and Tyr564 (Xiao et al., 2010). SHP-1-Tyr564 phosphorylation by Lyn is indispensable for maximal phosphatase activity, whereas SHP-1-Tyr536 can be phosphorylated by Lyn and another kinase(s) and this phosphorylation is necessary for efficient substrate recognition. A recent study suggests a novel concept for how SHP-1 may recognize its substrate kinases via a substrate recognition motif termed kinase tyrosine-based inhibitory motif (KTIM) (Abu-Dayyeh et al., 2008). Abu-Dayyeh et al. identified a number of KTIM ((I/V/L/S)-x-Y-x-x-(L/ V))-containing potential SHP-1 substrates that are evolutionarily conserved (Abu-Dayyeh et al., 2010). Thus, it is interesting to study the role of the KTIM motif in the recognition and dephosphorylation of the substrates, particularly Lyn, by SHP-1, since Lyn is the only SFK member that contains a KTIM (Abu-
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A SHP-1
Lyn* PLC- β3 SPS complex associated with Fc RI
B
Lyn*
SHP-1 PLC- β3
SHIP
Dok1/2
RasGAP
Ras Raf
Fc εRI ( β,γ ) MAPKs (ERK*, JNK*, p38*) other substrates
MEK ERK*
Fig. 2. Model for PLC-b3-mediated mutual regulation of Lyn and SHP-1 in FcεRI signaling. (A) Lyn activity is negatively regulated by SHP-1 in a PLC-b3-dependent manner (Xiao et al., 2011). Lyn and SHP-1 are associated with PLC-b3 (Xiao et al., 2010; Xiao et al., 2009) and FcεRI stimulation enhances these associations. PLC-b3 (Xiao et al., 2011), Lyn (Eiseman and Bolen, 1992), and SHP-1 (Kimura et al., 1997) are physically associated with FcεRI. Following FcεRI crosslinking, activated Lyn phosphorylates SHIP (Hernandez-Hansen et al., 2004; Hibbs et al., 2002; Xiao et al., 2005), which then recruits Dok1/2 to activate RasGAP. RasGAP negatively regulates the Ras/ERK cascade (Abramson et al., 2003; Mashima et al., 2009), accounting for negative regulation of FcεRI signaling by Lyn. Juxtaposition of Lyn and SHP-1 via their association with PLC-b3 promotes Lyn dephosphorylation at Tyr396 and inactivation, resulting in reversal of the Lyn-dependent inhibitory signals. (B) PLC-b3 augments the ability of Lyn to phosphorylate and activate SHP-1 (Xiao et al., 2010). SHP-1 dephosphorylates FcεRI (b, g), MAPKs, and other substrates, leading to termination of FcεRI signaling. Asterisk indicates the presence of KTIM. This model does not exclude the role of dual-specificity phosphatases in dephosphorylation of MAPKs (Salojin and Oravecz, 2007). Stat5 is also involved in FcεRI signaling (Barnstein et al., 2006). However, ERK activity is not affected by Stat5 deficiency (Barnstein et al., 2006).
Dayyeh et al., 2010). The data thus far obtained indicate that PLC-b3 provides an adaptor function to ensure efficient enzymatic activity of SHP-1 and to regulate its activity by Lyn. Therefore, with an additional component (i.e., Lyn) to the original SPS complex, we propose to rename this complex, without changing the name of the SPS complex, the Signaling Platform for SHP-1 activity. Does one see similar phosphatase-regulatory functions in other PLC-b isoforms (PLC-b1, -2, and -4)? This is unlikely, since the level of similarly is low at the C-terminal region of PLC-b isoforms. Our experiments showed that PLC-b2 does not interact with Stat5 or SHP-1. Interestingly, amino acid sequences similar to that of b11 were found in protozoan parasites and bacteria. These sequences might represent an immune evasion strategy. In addition to development of various tumors, deficiency in PLC-b3 affects the growth of various myeloid and lymphoid cells including mast cells, thus implicated in various aspects of innate and adaptive immunity. Summary Our recent studies have revealed an adaptor function of PLC-b3 through SPS complex formation of its C-terminal region (PLC-b3-CT) with SHP-1, Lyn, and Stat5. The platform provided by PLC-b3-CT promotes mutual regulation of Lyn and SHP-1 activities through phosphorylation of SHP-1-Tyr536 and SHP-1-Tyr564 and dephosphorylation of Lyn-Tyr396 as well as optimal dephosphorylation of Stat5Tyr694 by SHP-1. Lack of PLC-b3 or Lyn genes and loss-of-function mutation of SHP-1, which lead to reduced or abrogated activity of SHP-1, culminate in the development of MPN (and other tumors). The SPS complex regulates not only the proliferation, survival, and myeloid differentiation of HSCs, but also activation of mast cells through FcεRI. Conflict of interest No authors have conflicts of interest in this study.
Acknowledgments This study was supported in part by the MPN Foundation.
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Tsui HW, Siminovitch KA, de Souza L, Tsui FW. Motheaten and viable motheaten mice have mutations in the haematopoietic cell phosphatase gene. Nat Genet 1993;4:124–9. Uchida T, Matozaki T, Noguchi T, Yamao T, Horita K, Suzuki T, et al. Insulin stimulates the phosphorylation of Tyr538 and the catalytic activity of PTP1C, a protein tyrosine phosphatase with Src homology-2 domains. J Biol Chem 1994;269:12220–8. Waldo GL, Ricks TK, Hicks SN, Cheever ML, Kawano T, Tsuboi K, et al. Kinetic scaffolding mediated by a phospholipase C-b and Gq signaling complex. Science 2010;330:974–80. Wershil BK, Wang ZS, Gordon JR, Galli SJ. Recruitment of neutrophils during IgE-dependent cutaneous late phase reactions in the mouse is mast cell-dependent. partial inhibition of the reaction with antiserum against tumor necrosis factor-alpha. J Clin Invest 1991;87:446–53. Xiao W, Ando T, Wang HY, Kawakami Y, Kawakami T. Lyn- and PLC-b3-dependent regulation of SHP-1 phosphorylation controls Stat5 activity and myelomonocytic leukemia-like disease. Blood 2010;116:6003–13. Xiao W, Hong H, Kawakami Y, Kato Y, Wu D, Yasudo H, et al. Tumor suppression by phospholipase C-b3 via SHP-1-mediated dephosphorylation of Stat5. Cancer Cell 2009;16:161–71. Xiao W, Hong H, Kawakami Y, Lowell CA, Kawakami T. Regulation of myeloproliferation and M2 macrophage programming in mice by Lyn/Hck, SHIP, and Stat5. J Clin Invest 2008;118:924–34. Xiao W, Kashiwakura J, Hong H, Yasudo H, Ando T, Maeda-Yamamoto M, et al. Phospholipase C-b3 regulates FcεRI-mediated mast cell activation by recruiting the protein phosphatase SHP-1. Immunity 2011;34:893–904. Xiao W, Nishimoto H, Hong H, Kitaura J, Nunomura S, Maeda-Yamamoto M, et al. Positive and negative regulation of mast cell activation by Lyn via the FcεRI. J Immunol 2005;175:6885–92. Yang J, Liu L, He D, Song X, Liang X, Zhao ZJ, et al. Crystal structure of human protein-tyrosine phosphatase SHP-1. J Biol Chem 2003;278:6516–20. Yasudo H, Ando T, Xiao W, Kawakami Y, Kawakami T. Short Stat5-interacting peptide derived from phospholipase C-beta3 inhibits hematopoietic cell proliferation and myeloid differentiation. PLoS ONE 2011;6:e24995.
Contents lists available at SciVerse ScienceDirect
Advances in Enzyme Regulation journal homepage: www.elsevier.com/locate/ advenzreg
Regulation of proliferation, survival, differentiation, and activation by the Signaling Platform for SHP-1 phosphatase Toshiaki Kawakami*, Wenbin Xiao 1, Hiroki Yasudo, Yuko Kawakami Division of Cell Biology, La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037, USA
Introduction The recognition of a substrate by an enzyme has been taught as being very specific like ‘lock and key’ in a textbook of biochemistry. However, we now know that an enzyme can recognize various substrates. For example, in cell-free reactions, many protein-tyrosine kinases can phosphorylate different substrates ranging from an authentic in vivo substrate to artificial peptides such as poly(Glu– Tyr). Protein phosphatases can also dephosphorylate non-specific substrates such as p-nitrophenyl phosphate. The loose specificity in enzyme-substrate recognition would potentially create serious problems in many physiological processes such as metabolism, transcription, cell cycle progression, survival/apoptosis, differentiation, cytoskeletal rearrangement/cell movement, etc. However, mechanisms to enhance the specificity in enzyme-substrate recognition have evolved in the signal transduction pathways underpinning these functions. Some enzymes might have extremely high specificity, similar to that of antibody recognition of antigen. However, this strategy that simply increases the specificity might not be sufficient to ensure the efficient, fail-safe flow of information in signal transduction within a cell. Another strategy developed in evolution is restricting the enzyme-substrate interaction using an adaptor molecule. The prototypical example of this mechanism is found in the mitogen-activated protein kinase (MAPK) cascade: MAPK modules are composed of a cascade of three intracellular protein kinases (MKKK, MKK and MAPK) which are activated successively by phosphorylation events (Brunet and Pouyssegur, 1997; Burack and Shaw, 2000). In addition to the molecular selectivity of each enzyme for its substrate, exogenous proteins, such as the yeast Ste5 protein, serve as ‘chaperone’ proteins to tether all the members of a module and restrict signal transduction to this module. In addition to the efficient and accurate information flow, the adaptor-aided signaling cassette can direct the location and timing of enzymatic reactions, two factors essential for signal transduction. The human genome contains 518 genes encoding protein kinases (Manning et al., 2002) and 107 genes encoding protein-tyrosine phosphatases (Alonso et al., 2004). The SH2 domain-containing
* Corresponding author. E-mail address: [email protected] (T. Kawakami). 1 Present address: Department of Pathology, University Hospitals Case Medical Center, Case Western Reserve University, Cleveland, OH 44106, USA. 0065-2571/$ – see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.advenzreg.2011.09.003
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protein phosphatase 1 (SHP-1) is a protein-tyrosine phosphatase expressed in hematopoietic cells. SHP-1 has two tandem SH2 domains at the N-terminus, followed by a catalytic domain, and an inhibitory C-terminus. SHP-1 has a negative impact on lymphocyte signaling (Tsui et al., 2006). By contrast, its cousin SHP-2, which is widely expressed, in general promotes signaling pathways that lead to differentiation, cell growth, and migration (Lorenz, 2009; Poole and Jones, 2005; Tartaglia et al., 2004). In this review, we will summarize recent studies on how SHP-1 activity is regulated through formation of multimolecular complexes. Phospholipase C-b3 is a novel tumor suppressor Phospholipase C (PLC)-b is a small family of four enzymes that can produce diacylglycerol and inositol 1,4,5-trisphosphate (IP3) downstream of heterotrimeric G proteins (Rhee, 2001). As diacylglycerol can activate several isoforms of protein kinase C (PKC) (Nishizuka, 1992), and IP3 can mobilize Ca2þ (Berridge, 1993), PLC-b is implicated in promoting cell proliferation. PLC-b directly interacts with GTP-bound Ga subunits, leading to its catalytic activation. A recent study identified three Gaq-binding regions in PLC-b3 (Waldo et al., 2010): the short helix-turn-helix (Ha1 and Ha2) region following the C2 domain and the loop between the catalytic TIM barrel and C2 domain are critical for PLC catalytic activity; the third Gaq-interacting region, EF hands 3 and 4, are critical for GTP hydrolysis by Gaq. PLC-b2 and PLC-b3 can also be activated by bg subunits of the Gai/o family of G proteins (Camps et al., 1992; Katz et al., 1992; Lee et al., 1993). Consistent with their roles in G protein-coupled receptor signaling, chemokine-induced IP3 production, Ca2þ signaling, and migration are reduced in PLC-b2/ and PLC-b2/; PLC-b3/ neutrophils (Li et al., 2000) and T cells (Bach et al., 2007). The role of PLC-b3 in tumorigenesis has not been noticed until recently. PLC-b3 deficient mice die prematurely, mostly (w90%) by developing myeloproliferative neoplasm (MPN), which evolves to accelerated and blast-crisis stages. Some mice develop other tumors such as T cell lymphoma, carcinomas of skin and lung, and hamartoma (Xiao et al., 2009). This phenomenon is unique to PLC-b3/ mice, as PLCb2/ mice do not develop tumors or die prematurely. Aged PLC-b3/ mice with splenomegaly typically have increased numbers of c-KitþSca-1þLineage cells (KSL cells; enriched for hematopoietic stem cells [HSCs]), granulocyte-macrophage progenitors, and megakaryocyte-erythroid progenitors in the bone marrow (BM) and spleens. Consistent with these immunophenotype data, PLC-b3/ BM cells and splenocytes give rise to greater numbers of myeloid colonies than WT cells in methylcellulose medium, suggesting that PLC-b3/ HSCs and myeloid progenitors have an increased predisposition to differentiate into granulocytes. Moreover, PLC-b3/ BM and KSL cells are hypersensitive to cytokines such as GM-CSF and IL-3, a hallmark of human MPNs (Emanuel et al., 1991), and form macrophage and granulocytemacrophage colonies in the absence of growth factors, a feature characteristic of transformed cells. In vivo BrdU incorporation, in vitro cultures, and cell cycle analysis of KSL cells demonstrate increased proliferation in PLC-b3/ HSC-enriched populations (Xiao et al., 2009). Similarly, siRNAmediated silencing of PLC-b3 leads to enhanced proliferation in vascular endothelial growth factorstimulated endothelial cells (Bhattacharya et al., 2009). Apoptosis is less abundant in PLC-b3/ KSL cells. The MPN is transplantable with long-term HSC-enriched CD34 KSL cells derived from PLC-b3/ mice, indicating that the leukemic stem cells responsible for the development of MPD in PLC-b3/ mice are present in CD34 KSL cells. An interesting question is whether the microenviroment plays any role in the development of MPN in PLC-b3/ mice. The long latency of MPN in these mice probably is related to the microenviroment. However, when wild-type (wt) BM or HSCs were transferred into irradiated PLC-b3/ mice, no MPN was observed, suggesting that the microeviroment in PLC-b3/ mice is not sufficient for MPN development. Flow cytometry and immunofluorescence microscopy show that Stat5-Tyr694 phosphorylation is constitutively increased and localized in the nuclei of PLC-b3/ KSL cells. By contrast, Stat3 phosphorylation is comparable in PLC-b3/ and WT cells. Lymphomas and skin carcinoma from PLC-b3/ mice also exhibit increased phosphorylation of Stat5. Importantly, introduction of dominant-negative (DN) Stat5 into PLC-b3/ KSL cells suppresses their in vitro expansion, colony-forming ability, and MPN induction in transplanted mice. These results demonstrate that the increased Stat5 activity is responsible for the development of MPN (and probably other tumors as well) in PLC-b3/ mice. Thus, PLC-b3 is a novel tumor suppressor (Xiao et al., 2009).
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SPS complex The findings that PLC-b3 can suppress Stat5 activity were unexpected, as PLC-b3 is conventionally thought to be a component of GPCR signaling pathway. Structure-function analysis showed that the Cterminal portion of PLC-b3 (PLC-b3-CT, positions 809–1234) has the growth-inhibitory activity in IL-3dependent mouse Ba/F3 cells. Remarkably, we discovered a novel multimolecular complex termed SPS complex, which is composed of SHP-1, PLC-b3, and Stat5 (a transcription factor essential for proliferation and survival of myeloid cells) (Xiao et al., 2009). SHP-1 and Stat5 interact with PLC-b3 via the PLCb3-CT. The physical proximity of SHP-1 (enzyme) and Stat5 (substrate) brought about by these interactions allows an efficient dephosphorylation of Stat5 at Tyr496, the critical site of phosphorylation for Stat5 activation (Gouilleux et al., 1994). Since more than 80% of the three molecules can be found in the SPS complex after 5 min stimulation of Ba/F3 cells with IL-3, the Stat5-Tyr496 dephosphorylation is considered a major mechanism to keep Stat5 activation in check. Deficiency of PLC-b3 in knockout mice (Xiao et al., 2009) or loss-of-function mutation of SHP-1 in viable motheaten mice (Tsui et al., 1993) loses this mechanism to allow constitutive activation of Stat5 and MPN development. Interestingly, SHP-1 does not appear to dephosphorylate STAT3 in some types of leukemia cells (McKenzie et al., 2011), which may explain unchanged phosphorylation status of Stat3 in PLC-b3/ cells. PLC-b3 haploinsufficiency cooperates with c-myc for malignant transformation Haploinsufficiency is a typical feature of many classic tumor suppressors. Since we found that c-myc can induce in vitro transformation in PLC-b3þ/ and PLC-3/ mouse embryonic fibroblasts, we investigated whether PLC-b3 deficiency can cooperate with c-myc to induce in vivo tumor formation. Compared to Em-myc transgenic mice that develop B cell-lineage lymphomas with a long latency (Adams et al., 1985), pre-B cell lymphoma formation in Em-myc;PLC-b3þ/ mice was dramatically accelerated. These Em-myc;PLC-3þ/ lymphomas and PLC-b3/ lymphomas showed high levels of Stat5 phosphorylation. Importantly, all analyzed PLC-b3/ lymphomas showed as high c-myc expression as did Em-myc;PLC-b3þ/ lymphomas. Retroviral expression of DN Stat5 or PLC-b3-CT in Em-myc;PLC-3þ/ lymphoma cells suppressed their in vitro growth and colony formation, indicating that PLC-b3 haploinsufficiency cooperates with c-myc to transform fibroblasts and lymphocytes. Translocations of c-myc to immunoglobulin or other gene loci and thus abnormal expression of c-myc are causally linked to Burkitt’s lymphoma (Boxer and Dang, 2001). Interestingly, two of six Burkitt’s lymphoma cell lines tested, i.e., Daudi and Raji, exhibited very low levels of PLC-b3 protein and high levels of STAT5 phosphorylation. Overexpression of PLC-b3-CT or DN Stat5 in these cells blocked their growth and reduced STAT5 phosphorylation. These results suggest that reduced or abrogated expression of PLC-b3 can cooperate with active c-myc to induce lymphoma in mice and humans. Interestingly, approximately 10% percent of chronic lymphocytic leukemia samples showed low levels of PLC-b3 expression with high phospho-STAT5 levels. The results collectively suggest that reduced expression of PLC-b3 and thus the loss of the SHP-1-mediated Stat5 dephosphorylation mechanism cooperates with active c-myc to induce hematopoietic malignancies in mice and humans. LynL/L;PLC-b3L/L mice develop severe myelodysplastic syndrome (MDS)/MPN Searching for further molecular mechanisms for the increased Stat5 activity, we found dramatically increased phosphorylation of Lyn (a Src family kinase prominently expressed in myeloid and B cells) at Tyr396 in the activation loop in PLC-b3/ BM cells. Surprisingly, doubly deficient Lyn/;PLC-b3/ mice all died within 10 months after birth, with severe macrophage infiltration in the lungs (Xiao et al., 2010), similar to Lyn/;hck/ (Xiao et al., 2008), SHIP/ (Nakamura et al., 2004; Rauh et al., 2005; Xiao et al., 2008) and mev/mev (Xiao et al., 2009) mice. Myeloid cell infiltration was also seen in liver and kidney of Lyn/;PLC-b3/ mice. Comprehensive phenotype analysis indicated that unlike PLCb3/ and Lyn/ mice, Lyn/;PLC-b3/ mice had MDS/MPN with monocytosis, similar to human chronic myelomonocytic leukemia (Emanuel, 2008). Transplantation experiments showed that mice that received BM cells derived from Lyn/;PLC-3/ mice developed severe MDS/MPN with increased
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monocytes, anemia and thrombocytopenia, recapitulating the phenotype of Lyn/;PLC-b3/ mice. These results demonstrate that the MDS/MPN in Lyn/;PLC-b3/ mice is largely BM cell-autonomous. Lyn/;PLC-b3/ mice had higher levels of HSCs than PLC-b3/ and Lyn/ mice, as expected from the more severe form of MPN/MDS in the former mice. Consistent with this phenotype, Lyn/ ;PLC-b3/ KSL cells grew 2–3-fold faster than PLC-b3/ and Lyn/ cells. Cell cycle analysis revealed more Lyn/ KSL cells in the S/G2/M phases but comparable numbers in the G0 phase compared to wt cells, indicating that Lyn is a negative regulator for the G1-S transition, but has no effects on the G0-G1 checkpoint. On the other hand, compared to wt cells, both PLC-b3/ and Lyn/ ;PLC-b3/ KSL cells cycled more frequently into G1 and S/G2/M phases and stayed less frequently in the G0 phase. These results suggest that PLC-b3 is essential for regulation of the G0–G1 transition. Under low concentrations of IL-3 or GM-CSF, wt KSL cells gave rise to small numbers of colonies, and colony-forming efficiencies were significantly higher in Lyn/ and PLC-b3/ KSL cells. As expected, Lyn/;PLC-b3/ KSL cells generated the highest number of colonies, in line with their highest Stat5 activity among the four genotypes. Similar to PLC-b3/ cells, Stat5-Tyr694 phosphorylation was increased in Lyn/;PLC-3/ KSL cells before and after cytokine stimulation. Transduction with DN Stat5 showed that Stat5 is essential for the survival of these cells and that Stat5 is required for MDS/ MPN development in Lyn/;PLC-b3/ mice. Both PLC-b3 and Lyn are involved in the regulation of SHP-1 phosphorylation at Tyr536 and Tyr564 The molecular basis for the regulation of SHP-1 activity has been incompletely understood (Poole and Jones, 2005; Tsui et al., 1993). The C-terminal phosphorylation sites have been mapped to Tyr536 and Tyr564 residues (Uchida et al., 1994) and putative tyrosine kinases that catalyze these phosphorylations include Lyn (Hibbs et al., 2002; Xiao et al., 2005), Lck (Lorenz et al., 1994), and Src (Frank et al., 2004). Using phospho-specific antibodies against Tyr536 and Tyr564, we found that Tyr536 phosphorylation is reduced in both Lyn/ and PLC-b3/ cells, and is abolished in Lyn/;PLC-b3/ cells (Xiao et al., 2010). On the other hand, Tyr564 phosphorylation was not affected in PLC-b3/ cells, but reduced or almost abrogated in Lyn/ and Lyn/;PLC-b3/ cells. In vitro kinase assays confirmed that Lyn can phosphorylate SHP-1 at Tyr564 and Tyr536. These data demonstrate that Lyn is the predominant kinase that phosphorylates Tyr564, and PLC-b3 may help tyrosine kinases including Lyn to phosphorylate Tyr536 (Fig. 1). PLC-b3-CT interacts not only with SHP-1 (Xiao et al., 2009), but also with Lyn (Xiao et al., 2010) in Ba/F3 cells, splenocytes, and mast cells. Similar to PLC-b3/SHP-1 interactions, PLC-b3/Lyn interactions were enhanced by IL-3 stimulation. Furthermore, co-immunoprecipitation of Lyn with SHP-1 was also observed in wt splenocytes and PLC-b3 CT-overexpressing PLC-b3/ mast cells. Lyn/SHP-1 interactions were also enhanced by IL-3 stimulation. Interestingly, Lyn/SHP-1 interactions were only weakly detected in PLC-b3/ splenocytes and BMMCs, suggesting that PLC-b3 CT facilitates the interaction between Lyn and SHP-1. Thus, these results added a new member, i.e., Lyn, to the SPS complex (Xiao et al., 2010). Roles of SHP-1 Tyr564 and Tyr536 phosphorylation To investigate the effect of Tyr536 and Tyr564 phosphorylation on SHP-1 phosphatase activity, wt, Y536F, Y564F or doubly YF mutant (DYF) SHP-1 vectors were introduced into mev/mev CD34KSL cells. As expected, wt SHP-1 increased a phosphatase activity by 4-fold over empty vector control. Y536F mutant exhibited a phosphatase activity similar to wt enzyme. By contrast, Y564F and DYF mutants had activities similar to vector control, suggesting that Tyr564 phosphorylation is critical for SHP-1 phosphatase activity. Moreover, we found that SHP-1 phosphatase activity is inversely correlated with Stat5 activity, in vitro cell growth, and MPN-causing abilities of HSCs. Despite the normal catalytic activity, Y536F SHP-1 exhibited lower suppression of Stat5 phosphorylation and in vitro cell growth in mev/mev HSCs and mast cells, compared to wt SHP-1. This could be due to weaker interactions between Y536F SHP-1 and Stat5. Indeed, co-immunoprecipitation of SHP-1 with Stat5 was drastically reduced in mev/mev mast cells expressing Y536F or DYF SHP-1. By
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A SH2
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Fig. 1. A model for SHP-1 activation and recognition of its substrate Stat5. (A) SHP-1 is phosphorylated at Tyr564 predominantly by Lyn and at Tyr536 by Lyn and other kinase(s) including Jak2. Inactive SHP-1 (Top left) has a closed conformation with the catalytic site of the PTP domain capped by the N-SH2 domain (Yang et al., 2003). The Tyr536 phosphorylation site of SHP-1 interacts with Stat5 most probably via the SH2 domain of the latter molecule (Bottom). The Tyr564 phosphorylation site of SHP-1 interacts with N-SH2 to render the enzyme active. N-terminal ends of SHP-1 and Stat5 proteins are shown by ‘N’. (B) SHP-1 and Lyn interact with PLC-b3-CT (Xiao et al., 2010; Xiao et al., 2009). Tyr694 phosphorylated Stat5 is recruited to the PLC-b3-nucleated SPS complex containing Lyn and SHP-1, and dephosphorylated by an active SHP-1 enzyme that is phosphorylated at Tyr536 and Tyr564. Stat5 dimer is depicted as a monomer for simplicity.
contrast, wt and Y564F SHP-1 were well co-immunoprecipitated with Stat5. These results suggest that SHP-1 phosphorylation of Tyr536 is critical for interaction with Stat5. Consistent with our observation that Lyn phosphorylates SHP-1 at Tyr564, SHP-1 phosphatase activity was reduced by 40% in Lyn/ mast cells. Lyn/;PLC-b3/ cells had a very low level of SHP-1 phosphatase activity, similar to mev/mev cells (which had 10–20% activity of wt enzyme), showing that the reduced SHP-1 phosphatase activity is a common signaling defect shared by mev/mev and Lyn/;PLCb3/ mice. Although overexpression of wt SHP-1 suppressed the in vitro growth of mev/mev CD34KSL cells as well as wt, Lyn/, and PLC-b3/ cells, it had little effect on Lyn/;PLC-b3/ cells. Wt SHP-1overexpressing CD34KSL cells from Lyn/;PLC-b3/ mice still caused MPN upon transplantation. Consistent with these results, the transduced wt SHP-1 was not phosphorylated at Tyr536 or Tyr564 in Lyn/;PLC-b3/ cells. However, transduction of Lyn/;PLC-b3/ cells with SHP-1 with phosphomimetic Y536E/Y564E (DYE) mutations increased SHP-1 phosphatase activity by 10-fold in Lyn/;PLCb3/ cells, in contrast to a mere 2-fold increase by wt SHP-1 expression. Importantly, DYE-transduced cells exhibited substantially lower levels of Stat5 phosphorylation and in vitro growth rate and failed to exhibit MPN-causing properties. These results suggest that severely reduced SHP-1 phosphatase activity is responsible for the MDS/MPN-associated phenotypes observed in Lyn/;PLC-b3/ mice. Growth-inhibitory activity of short Stat5-binding peptides within PLC-b3-CT More recently, we finely mapped the Stat5- and SHP-1-binding sites within PLC-b3-CT, using affinity pulldown and a large panel of GST fusion proteins containing various portions of PLC-b3-CT
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(Yasudo et al., PLoS ONE, 2011). By designing the fusion constructs to narrow down the binding sites within less than 40 amino acid residues, we found that three noncontiguous regions, designated a (positions 917–943), b (positions 983–1000), and c (positions 1032–1069), bound to Stat5 and two regions, designated c (positions 1032–1069) and d (positions 1182–1209) bound to SHP-1. Given the critical importance of Stat5 for the proliferation, survival, and differentiation of HSCs in PLC-b3/ mice (Xiao et al., 2009), we tested whether overexpression of the Stat5-interacting, N-terminally myc-tagged peptides affects the in vitro growth of Ba/F3 cells. The experiments indicated that the Stat5-binding activity of peptides b and c may be critical for growth suppression. Because peptide b exhibited approximately 80% potency of PLC-b3-CT in Ba/F3 cell growth inhibition, we tested even shorter peptides in this region. Among them, an 11-residue peptide (peptide b11; positions 988– 998) exhibited a growth-inhibitory activity as strong as >80% that of PLC-b3-CT. As expected, Stat5 phosphorylation at Tyr694 was significantly reduced in peptide b11-expressing cells. The relevance of these in vitro studies was verified by reduced myeloid colony-forming activity of the peptide b11expressing cells and by the failure of peptide b11-expressing lin BM cells derived from PLC-b3/ mice to cause MPN development upon transplantation. PLC-b3 regulates FceRI-mediated mast cell activation Is PLC-b3-mediated regulation of SHP-1 activity relevant to other biological events? We studied this point in mast cell activation (Xiao et al., 2011). Mast cells are major effectors in high-affinity IgE receptor (FcεRI)-dependent allergic reactions (Kawakami and Galli, 2002). Activated mast cells secrete preformed proinflammatory mediators (e.g., histamine, proteases, proteoglycans, and nucleotides) as well as de novo synthesized lipids (e.g., leukotrienes and prostaglandins) and polypeptides (e.g., cytokines and chemokines). These substances lead to the development of allergic inflammation. Antigen challenge in IgE-sensitized mice induces life-threatening allergic reactions termed anaphylaxis through FcεRI-mediated mast cell activation (Inagaki et al., 1986; Wershil et al., 1991). PLC-b3/ mice showed blunted late-phase, but not acute-phase, anaphylactic responses. Consistent with this, FcεRI stimulation of PLC-b3/ mast cells exhibited substantially reduced cytokine production, but normal degranulation. Reduced cytokine production in PLC-b3/ cells could be accounted for by increased activity of the negative regulator Lyn and reduced activities of the positive regulators MAPKs. Mechanistically, PLC-b3 constitutively interacts with FcεRI, Lyn, and SHP-1. SHP-1 likely recognizes its substrates Lyn and MAPKs via the recently described kinase tyrosine-based inhibitory motif, KTIM (Abu-Dayyeh et al., 2008). Consistent with PLC-b3/SHP-1-mediated repression of Lyn kinase activity by dephosphorylation at Tyr396, FcεRI-mediated phenotypes were similar in PLC-b3/ and SHP-1-mutant mev/mev mast cells. In PLC-b3/ mast cells, Lyn is constitutively active and negatively regulates FcεRImediated activation of the lipid phosphatase SHIP-1 (Hernandez-Hansen et al., 2004; Xiao et al., 2005); phosphorylated SHIP-1 recruits Dok adaptors to activate RasGAP (Abramson et al., 2003); RasGAP negatively regulates the Ras/Raf/MEK/ERKs pathway (Mashima et al., 2009). Thus, this study defined the PLC-b3/SHP-1-mediated novel signaling pathway (Fig. 2) for FcεRI-mediated cytokine production (Xiao et al., 2011). Future perspectives Information flow between SHP-1 and Lyn is bidirectional, since SHP-1 dephosphorylates Lyn-Tyr396 and Lyn phosphorylates SHP-1 at two residues, Tyr536 and Tyr564 (Xiao et al., 2010). SHP-1-Tyr564 phosphorylation by Lyn is indispensable for maximal phosphatase activity, whereas SHP-1-Tyr536 can be phosphorylated by Lyn and another kinase(s) and this phosphorylation is necessary for efficient substrate recognition. A recent study suggests a novel concept for how SHP-1 may recognize its substrate kinases via a substrate recognition motif termed kinase tyrosine-based inhibitory motif (KTIM) (Abu-Dayyeh et al., 2008). Abu-Dayyeh et al. identified a number of KTIM ((I/V/L/S)-x-Y-x-x-(L/ V))-containing potential SHP-1 substrates that are evolutionarily conserved (Abu-Dayyeh et al., 2010). Thus, it is interesting to study the role of the KTIM motif in the recognition and dephosphorylation of the substrates, particularly Lyn, by SHP-1, since Lyn is the only SFK member that contains a KTIM (Abu-
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A SHP-1
Lyn* PLC- β3 SPS complex associated with Fc RI
B
Lyn*
SHP-1 PLC- β3
SHIP
Dok1/2
RasGAP
Ras Raf
Fc εRI ( β,γ ) MAPKs (ERK*, JNK*, p38*) other substrates
MEK ERK*
Fig. 2. Model for PLC-b3-mediated mutual regulation of Lyn and SHP-1 in FcεRI signaling. (A) Lyn activity is negatively regulated by SHP-1 in a PLC-b3-dependent manner (Xiao et al., 2011). Lyn and SHP-1 are associated with PLC-b3 (Xiao et al., 2010; Xiao et al., 2009) and FcεRI stimulation enhances these associations. PLC-b3 (Xiao et al., 2011), Lyn (Eiseman and Bolen, 1992), and SHP-1 (Kimura et al., 1997) are physically associated with FcεRI. Following FcεRI crosslinking, activated Lyn phosphorylates SHIP (Hernandez-Hansen et al., 2004; Hibbs et al., 2002; Xiao et al., 2005), which then recruits Dok1/2 to activate RasGAP. RasGAP negatively regulates the Ras/ERK cascade (Abramson et al., 2003; Mashima et al., 2009), accounting for negative regulation of FcεRI signaling by Lyn. Juxtaposition of Lyn and SHP-1 via their association with PLC-b3 promotes Lyn dephosphorylation at Tyr396 and inactivation, resulting in reversal of the Lyn-dependent inhibitory signals. (B) PLC-b3 augments the ability of Lyn to phosphorylate and activate SHP-1 (Xiao et al., 2010). SHP-1 dephosphorylates FcεRI (b, g), MAPKs, and other substrates, leading to termination of FcεRI signaling. Asterisk indicates the presence of KTIM. This model does not exclude the role of dual-specificity phosphatases in dephosphorylation of MAPKs (Salojin and Oravecz, 2007). Stat5 is also involved in FcεRI signaling (Barnstein et al., 2006). However, ERK activity is not affected by Stat5 deficiency (Barnstein et al., 2006).
Dayyeh et al., 2010). The data thus far obtained indicate that PLC-b3 provides an adaptor function to ensure efficient enzymatic activity of SHP-1 and to regulate its activity by Lyn. Therefore, with an additional component (i.e., Lyn) to the original SPS complex, we propose to rename this complex, without changing the name of the SPS complex, the Signaling Platform for SHP-1 activity. Does one see similar phosphatase-regulatory functions in other PLC-b isoforms (PLC-b1, -2, and -4)? This is unlikely, since the level of similarly is low at the C-terminal region of PLC-b isoforms. Our experiments showed that PLC-b2 does not interact with Stat5 or SHP-1. Interestingly, amino acid sequences similar to that of b11 were found in protozoan parasites and bacteria. These sequences might represent an immune evasion strategy. In addition to development of various tumors, deficiency in PLC-b3 affects the growth of various myeloid and lymphoid cells including mast cells, thus implicated in various aspects of innate and adaptive immunity. Summary Our recent studies have revealed an adaptor function of PLC-b3 through SPS complex formation of its C-terminal region (PLC-b3-CT) with SHP-1, Lyn, and Stat5. The platform provided by PLC-b3-CT promotes mutual regulation of Lyn and SHP-1 activities through phosphorylation of SHP-1-Tyr536 and SHP-1-Tyr564 and dephosphorylation of Lyn-Tyr396 as well as optimal dephosphorylation of Stat5Tyr694 by SHP-1. Lack of PLC-b3 or Lyn genes and loss-of-function mutation of SHP-1, which lead to reduced or abrogated activity of SHP-1, culminate in the development of MPN (and other tumors). The SPS complex regulates not only the proliferation, survival, and myeloid differentiation of HSCs, but also activation of mast cells through FcεRI. Conflict of interest No authors have conflicts of interest in this study.
Acknowledgments This study was supported in part by the MPN Foundation.
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