Hematopoietic Lineage Cell-specific Protein Research Articles

Human Cytomegalovirus Upregulates Expression of HCLS1 Resulting in Increased Cell Motility and Transendothelial Migration during Latency.

SummaryHuman cytomegalovirus establishes a lifelong, latent infection in the human host and can cause significant morbidity and mortality, particularly, in immunocompromised individuals. One established site of HCMV latency and reactivation is in cells of the myeloid lineage. In undifferentiated myeloid cells, such as CD14+ monocytes, virus is maintained latently. We have recently reported an analysis of the total proteome of latently infected CD14+ monocytes, which identified an increase in hematopoietic lineage cell-specific protein (HCLS1). Here we show that this latency-associated upregulation of HCLS1 occurs in a US28-dependent manner and stabilizes actin structure in latently infected cells. This results in their increased motility and ability to transit endothelial cell layers. Thus, latency-associated increases in monocyte motility could aid dissemination of the latently infected reservoir, and targeting this increased motility could have an impact on the ability of latently infected monocytes to distribute to tissue sites of reactivation.

Hematopoietic lineage cell-specific protein 1 (HS1), a hidden player in migration, invasion, and tumor formation, is over-expressed in ovarian carcinoma cells.

Hematopoietic lineage cell-specific protein 1 (HS1), which is the hematopoietic homolog of cortactin, is an actin-binding protein and Lyn substrate. It is upregulated in several cancers and its expression level is associated with increased cell migration, metastasis, and poor prognosis. Here we investigated the expression and roles of HS1 in ovarian carcinoma cells. We analyzed the expression of HS1 in 171 ovarian cancer specimens and determined the association between HS1 expression and clinicopathological characteristics, including patient outcomes. In patients with stage II–IV disease, positive HS1 expression was associated with significantly worse overall survival than negative expression (P < 0.05). HS1 was localized in invadopodia in some ovarian cancer cells and was required for invadopodia formation. Migration and invasion of ovarian cancer cells were suppressed by down-regulation of HS1, but increased in cells that over-expressed exogenous HS1. Furthermore, ovarian cancer cells that expressed HS1 shRNA exhibited reduced tumor formation in a mouse xenograft model. Finally, we found that tyrosine phosphorylation of HS1 was essential for cell migration and invasion. These findings show that HS1 is a useful biomarker for the prognosis of patients with ovarian carcinoma and is a critical regulator of cytoskeleton remodeling involved in cell migration and invasion.

Hematopoietic lineage cell-specific protein 1 immunoreactivity indicates an increased risk of poor overall survival in patients with ovarian carcinoma.

Hematopoietic lineage cell-specific protein 1 (HS1) is a 75-kDa intracellular protein that is expressed primarily in hematopoietic cells. Several previous studies have demonstrated the association between HS1 expression and a poor prognosis in hematopoietic malignancies; however, in solid tumors, no studies not been reported. The present study examined the distribution and expression of HS1 in human epithelial ovarian carcinoma (EOC) to determine its clinical significance. Paraffin sections were obtained from EOC tissues and immunostained with HS1 antibody, and then the staining intensities were evaluated. Overall survival (OS) was determined using the Kaplan-Meier estimator method, and multivariate analysis was performed using the Cox proportional hazards analysis. In total, 195 patients with EOC (median age, 56 years) were enrolled into the present study. HS1 immunoreactivity was categorized based on expression levels: Low (89/195; 45.6%) and high (106/195; 54.4%). Results demonstrated no association between expression level(s) and any clinicopathological parameter including age, International Federation of Gynecology and Obstetrics (FIGO) staging, type of chemotherapy or type of surgery received. The 5-year OS rates of patients who demonstrated low (n=89) and high (n=106) HS1 expression were 90.4 and 66.7%, respectively. The OS times for patients with high HS1 expression were significantly shorter compared with those for patients exhibiting low HS1 expression (P=0.0065). Results obtained from the multivariate analysis demonstrated that the FIGO stage and the amount of HS1 expressed were significant independent prognostic markers for poorer OS (hazard ratio, 3.539; 95% confidence interval, 1.221-12.811; P=0.0187). High HS1 expression levels may serve as a useful biomarker in patients with EOC who are likely to exhibit an unfavorable clinical outcome.

Durable and Specific Inhibition of ROR1 Signaling Associates with Prolonged Progression Free Survival in Patients with Chronic Lymphocytic Leukemia Treated with Cirmtuzumab

Small-molecule drugs that inhibit survival signals in leukemia cells have demonstrated high efficacy in the treatment of patients (pts) with chronic lymphocytic leukemia (CLL). Although not directly cytotoxic for leukemia cells, these drugs block key signaling pathways that govern leukemia-cell trafficking, proliferation, and survival. However, non-specific inhibition of enzymes involved in other signaling pathways in non-neoplastic cells may result in toxicity, which can limit the therapeutic index of such drugs, at least in some settings.Monoclonal antibodies (mAbs) also may be used as antagonists of signaling by interfering with receptor-ligand interactions or reducing the availability of cell-surface receptors. Unlike small-molecule inhibitors, mAbs may function as highly specific inhibitors of their target receptors and have superior pharmacokinetics (PK) due to a longer half-life. Based on this rationale, we developed cirmtuzumab, a humanized mAb specific for ROR1. ROR1 is an oncoembronic cell-surface receptor for Wnt5a, which induces non-canonical Wnt signaling, leading to Rho-GTPase activation and phosphorylation of hematopoietic lineage cell-specific protein 1 (HS1), and enhances leukemia-cell migration, survival, and proliferation. ROR1 is strongly expressed on CLL, but not on virtually all normal postpartum tissues. As such, ROR1 appears to be a selective target for anti-cancer therapy.We conducted a phase 1, dose-escalation trial in pts with relapsed/refractory CLL. Eligible pts had progressive disease with an indication for therapy, as per iwCLL guidelines. Pts received four biweekly infusions of cirmtuzumab at doses ranging from 0.015 to 20mg/kg. We performed PK studies on plasma samples using an ELISA assay to assess for cirmtuzumab binding to immobilized recombinant ROR1. Pharmacodynamic (PD) studies included analyses of leukemia-cell activation of Rho-GTPases and phosphorylation/activation of HS1.Twenty-five pts enrolled and received cirmtuzumab. Cirmtuzumab was safe and well-tolerated. There were no drug-related SAEs, DLTs, or infusion-related reactions. At higher doses, PK studies showed the half-life of cirmtuzumab at higher doses was 32.4 days (SD 1.9 days) and cirmtuzumab levels remained detectable in the plasma until approximately 3 months following the final infusion. No patient developed neutralizing autoantibodies, and no circulating ROR1 antigen was detected.Pharmacodynamic studies confirmed sustained target inhibition. At doses of 2 mg/kg or higher, we detected inhibition of leukemia-cell Rho-GTPase activation within 24 hours of the initial cirmtuzumab dose. Inhibition of leukemia-cell activation of Rho-GTPases was sustained for weeks after the final infusion until the plasma cirmtuzumab levels became negligible (<200 ng/mL). Similar findings were observed with phosphorylated HS-1.Clinical effects were in line with those of the PD and PK results. Despite limited treatment with just four doses of antibody, there were encouraging signs of clinical activity, including treatment-related reduction in circulating lymphocyte counts, lymph node size, and/or leukemia-cell infiltration of the marrow. Importantly, nearly all patients treated at dose levels ≥2 mg/kg had arrested disease progression, despite having progressive disease upon study entry. Moreover, 16 of 19 evaluable patients had stable disease at the time of response assessment (2 months following the final infusion per iwCLL guidelines). Patients did not require subsequent therapy for prolonged periods. The median time to requiring next treatment due to progressive disease was 259 days (95% CI 168-265 days). This also correlated with the time at which cirmtuzumab levels and PD effects became undetectable.Specific inhibition of ROR1 signaling by cirmtuzumab inhibits leukemia-cell RhoGTPase activation and HS-1 phosphorylation, and arrests disease progression without significant toxicity. This represents a novel approach to inhibiting growth/survival signaling in CLL, providing an advantageous PK and specific PD relative to that of small-molecule inhibitors, potentially ushering in a new era of immunopharmacology. DisclosuresChoi:Gilead, Genentech, Abbvie, and PCYC: Speakers Bureau; AbbVie, Genentech, and PCYC: Consultancy; AbbVie: Other: Institutional research funding. Kipps:Celgene: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Speakers Bureau; Oncternal: Research Funding; Pharmacyclics, LLC, an AbbVie Company: Consultancy, Honoraria; Roche: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria, Research Funding.

Suppressive effect of CORM-2 on LPS-induced platelet activation by glycoprotein mediated HS1 phosphorylation interference.

In recent years, it has been discovered that septic patients display coagulation abnormalities. Platelets play a major role in the coagulation system. Studies have confirmed that carbon monoxide (CO) has important cytoprotective and anti-inflammatory function. However, whether CO could alter abnormal activation of platelets and coagulation and thereby reduce the incidence of mortality during sepsis has not been defined. In this report, we have used CO-releasing molecules (CORM-2) to determine whether CO inhibits LPS-induced abnormal activation of platelets and have explored the potential mechanisms. LPS was used to induce activation of platelets in vitro, which were purified from the peripheral venous blood of healthy adult donors. CORM-2 was applied as a potential therapeutic agent. CORM-2 preconditioning and delayed treatment were also studied. We found that in the LPS groups, the function of platelets such as spreading, aggregation, and release were enhanced abnormally. By contrast, the platelets in the CORM-2 group were gently activated. Further studies showed that the expression of platelet membrane glycoproteins increased in the LPS group. Coincidently, both hematopoietic lineage cell-specific protein 1 and its phosphorylated form also increased dramatically. These phenomena were less dramatically seen in the CORM-2 groups. Taken together, we conclude that during LPS stimulation, platelets were abnormally activated, and this functional state may be associated with the signal that is transmitted between membrane glycoproteins and HS1. CORM-released CO suppresses the abnormal activation of platelets by interfering with glycoprotein-mediated HS1 phosphorylation.

Coordinate control of cytoskeletal remodeling and calcium mobilization during T‐cell activation

Ca(2+) mobilization and cytoskeletal reorganization are key hallmarks of T-cell activation, and their interdependence has long been recognized. Recent advances in the field have elucidated the molecular pathways that underlie these events and have revealed several points of intersection. Ca(2+) signaling can be divided into two phases: initial events leading to release of Ca(2+) from endoplasmic reticulum stores, and a second phase involving STIM 1 (stromal interaction molecule 1) clustering and CRAC (calcium release activated calcium) channel activation. Cytoskeletal dynamics promote both phases. During the first phase, the actin cytoskeleton promotes mechanotransduction and serves as a dynamic scaffold for microcluster assembly. Proteins that drive actin polymerization such as WASp (Wiskott-Aldrich syndrome protein) and HS1 (hematopoietic lineage cell-specific protein 1) promote signaling through PLCγ1 (phospholipase Cγ1) and release of Ca(2+) from endoplasmic reticulum stores. During the second phase, the WAVE (WASP-family verprolin homologous protein) complex and the microtubule cytoskeleton promote STIM 1 clustering at sites of plasma membrane apposition, opening Orai channels. In addition, gross cell shape changes and organelle movements buffer local Ca(2+) levels, leading to sustained Ca(2+) mobilization. Conversely, elevated intracellular Ca(2+) activates cytoskeletal remodeling. This can occur indirectly, via calpain activity, and directly, via Ca(2+) -dependent cytoskeletal regulatory proteins such as myosin II and L-plastin. While it is true that the cytoskeleton regulates Ca(2+) responses and vice versa, interdependence between Ca(2+) and the cytoskeleton also encompasses signaling events that occur in parallel, downstream of shared intermediates. Inositol cleavage by PLCγ1 simultaneously triggers both endoplasmic reticulum store release and diacylglycerol-dependent microtubule organizing center reorientation, while depleting the pool of phosphatidylinositol-4,5-bisphosphate, an activator of multiple actin-regulatory proteins. The close interdependence of Ca(2+) signaling and cytoskeletal dynamics in T cells provides positive feedback mechanisms for T-cell activation and allows for finely tuned responses to extracellular cues.

MDM2 promoter SNP309 mutation and HS1 expression in chronic lymphocytic leukemia patients ( exploring their assumed prognostic value)

Background Chronic lymphocytic leukemia (CLL) is one of the most common forms of leukemia and thus research is required to identify cost-effective and efficient prognostic markers. Aim The aim of the study was to identify the significance of MDM2 promoter SNP309 mutation and hematopoietic lineage cell-specific protein 1 (HS1) expression in the prognosis of CLL. Patients and methods Forty-eight newly diagnosed Egyptian CLL patients were clinically evaluated and assessed for MDM2 promoter SNP309 mutation, HS1 expression, ZAP-70 expression, 17p13 deletion, and 13q14 deletion. Thirty-one patients received chemotherapy, and the type of response to chemotherapy was observed. Results The results showed that MDM2 promoter SNP309 mutation was associated with cytogenetic abnormalities, the need to receive chemotherapy, and the response to fludarabine/cyclophosphamide in treated patients. HS1 expression was not associated with any of the studied CLL characteristics at diagnosis. Moreover, no association was found between HS1 expression and the need to receive chemotherapy during the study period. However, a significant association between HS1 expression and the response to fludarabine/cyclophosphamide was found. Conclusion The study concluded that, despite the opposing researchers’ reports, MDM2 promoter SNP309 mutation and HS1 expression are candidate prognostic markers for CLL patients awaiting a large-scale evaluation.

HS1, a Lyn kinase substrate, is abnormally expressed in B-chronic lymphocytic leukemia and correlates with response to fludarabine-based regimen.

In B-Chronic Lymphocytic Leukemia (B-CLL) kinase Lyn is overexpressed, active, abnormally distributed, and part of a cytosolic complex involving hematopoietic lineage cell-specific protein 1 (HS1). These aberrant properties of Lyn could partially explain leukemic cells’ defective apoptosis, directly or through its substrates, for example, HS1 that has been associated to apoptosis in different cell types. To verify the hypothesis of HS1 involvement in Lyn-mediated leukemic cell survival, we investigated HS1 protein in 71 untreated B-CLL patients and 26 healthy controls. We found HS1 overexpressed in leukemic as compared to normal B lymphocytes (1.38±0.54 vs 0.86±0.29, p<0.01), and when HS1 levels were correlated to clinical parameters we found a higher expression of HS1 in poor-prognosis patients. Moreover, HS1 levels significantly decreased in ex vivo leukemic cells of patients responding to a fludarabine-containing regimen. We also observed that HS1 is partially localized in the nucleus of neoplastic B cells. All these data add new information on HS1 study, hypothesizing a pivotal role of HS1 in Lyn-mediated modulation of leukemic cells’ survival and focusing, one more time, the attention on the BCR-Lyn axis as a putative target for new therapeutic strategies in this disorder.

The Actin Regulatory Protein HS1 Is Required for Antigen Uptake and Presentation by Dendritic Cells

The hematopoietic actin regulatory protein hematopoietic lineage cell-specific protein 1 (HS1) is required for cell spreading and signaling in lymphocytes, but the scope of HS1 function in Ag presentation has not been addressed. We show that dendritic cells (DCs) from HS1(-/-) mice differentiate normally and display normal LPS-induced upregulation of surface markers and cytokines. Consistent with their normal expression of MHC and costimulatory molecules, HS1(-/-) DCs present OVA peptide efficiently to CD4(+) T cells. However, presentation of OVA protein is defective. Similarly, MHC class I-dependent presentation of VSV8 peptide to CD8(+) T cells occurs normally, but cross-presentation of GRP94/VSV8 complexes is defective. Analysis of Ag uptake pathways shows that HS1 is required for receptor-mediated endocytosis, but not for phagocytosis or macropinocytosis. HS1 interacts with dynamin 2, a protein involved in scission of endocytic vesicles. However, HS1(-/-) DCs showed decreased numbers of endocytic invaginations, whereas dynamin-inhibited cells showed accumulation of these endocytic intermediates. Taken together, these studies show that HS1 promotes an early step in the endocytic pathway that is required for efficient Ag presentation of exogenous Ag by DCs.

Hematopoietic Lineage Cell-Specific Protein 1 Functions in Concert with the Wiskott–Aldrich Syndrome Protein To Promote Podosome Array Organization and Chemotaxis in Dendritic Cells

Dendritic cells (DCs) are professional APCs that reside in peripheral tissues and survey the body for pathogens. Upon activation by inflammatory signals, DCs undergo a maturation process and migrate to lymphoid organs, where they present pathogen-derived Ags to T cells. DC migration depends on tight regulation of the actin cytoskeleton to permit rapid adaptation to environmental cues. We investigated the role of hematopoietic lineage cell-specific protein 1 (HS1), the hematopoietic homolog of cortactin, in regulating the actin cytoskeleton of murine DCs. HS1 localized to lamellipodial protrusions and podosomes, actin-rich structures associated with adhesion and migration. DCs from HS1(-/-) mice showed aberrant lamellipodial dynamics. Moreover, although these cells formed recognizable podosomes, their podosome arrays were loosely packed and improperly localized within the cell. HS1 interacts with Wiskott-Aldrich syndrome protein (WASp), another key actin-regulatory protein, through mutual binding to WASp-interacting protein. Comparative analysis of DCs deficient for HS1, WASp or both proteins revealed unique roles for these proteins in regulating podosomes with WASp being essential for podosome formation and with HS1 ensuring efficient array organization. WASp recruitment to podosome cores was independent of HS1, whereas HS1 recruitment required Src homology 3 domain-dependent interactions with the WASp/WASp-interacting protein heterodimer. In migration assays, the phenotypes of HS1- and WASp-deficient DCs were related, but distinct. WASp(-/y) DCs migrating in a chemokine gradient showed a large decrease in velocity and diminished directional persistence. In contrast, HS1(-/-) DCs migrated faster than wild-type cells, but directional persistence was significantly reduced. These studies show that HS1 functions in concert with WASp to fine-tune DC cytoarchitecture and direct cell migration.

Deregulation of Mitochondrial Membrane Potential by Mitochondrial Insertion of Granzyme B and Direct Hax-1 Cleavage

The cytoplasm and the nucleus have been identified as activity sites for granzyme B (GrB) following its delivery from cytotoxic lymphocyte granules into target cells. Here we report on the ability of exogenous GrB to insert into and function within a proteinase K-resistant mitochondrial compartment. We identified Hax-1 (HS-1-associated protein X-1), a mitochondrial protein involved in the maintenance of mitochondrial membrane potential, as a GrB substrate within the mitochondrion. GrB cleaves Hax-1 into two major fragments: an N-terminal fragment that localizes to mitochondria and a C-terminal fragment that localizes to the cytosol after being released from GrB-treated mitochondria. The N-terminal Hax-1 fragment major cellular impact is on the regulation of mitochondrial polarization. Overexpression of wild-type Hax-1 or its uncleavable mutant form protects the mitochondria against GrB or valinomycin-mediated depolarization. The N-terminal Hax-1 fragment functions as a dominant negative form of Hax-1, mediating mitochondrial depolarization in a cyclophilin D-dependent manner. Thus, induced expression of the N-terminal Hax-1 fragment results in mitochondrial depolarization and subsequent lysosomal degradation of such altered mitochondria. This study is the first to demonstrate GrB activity within the mitochondrion and to identify Hax-1 cleavage as a novel mechanism for GrB-mediated mitochondrial depolarization.

Hematopoietic Lineage Cell-Specific Protein 1 Is Recruited to the Immunological Synapse by IL-2-Inducible T Cell Kinase and Regulates Phospholipase Cγ1 Microcluster Dynamics during T Cell Spreading

Productive T cell activation requires efficient reorganization of the actin cytoskeleton. We showed previously that the actin-regulatory protein, hematopoietic lineage cell-specific protein 1 (HS1), is required for the stabilization of F-actin and Vav1 at the immunological synapse and for efficient calcium responses. The Tec family kinase IL-2-inducible T cell kinase (Itk) regulates similar aspects of T cell activation, suggesting that these proteins act in the same pathway. Using video microscopy, we show that T cells lacking Itk or HS1 exhibited similar defects in actin responses, extending unstable lamellipodial protrusions upon TCR stimulation. HS1 and Itk could be coimmunoprecipitated from T cell lysates, and GST-pulldown studies showed that Itk's Src homology 2 domain binds directly to two phosphotyrosines in HS1. In the absence of Itk, or in T cells overexpressing an Itk Src homology 2 domain mutant, HS1 failed to localize to the immunological synapse, indicating that Itk serves to recruit HS1 to sites of TCR engagement. Because Itk is required for phospholipase C (PLC)gamma1 phosphorylation and calcium store release, we examined the calcium signaling pathway in HS1(-/-) T cells in greater detail. In response to TCR engagement, T cells lacking HS1 exhibited diminished calcium store release, but TCR-dependent PLCgamma1 phosphorylation was intact, indicating that HS1's role in calcium signaling is distinct from that of Itk. HS1-deficient T cells exhibited defective cytoskeletal association of PLCgamma1 and altered formation of PLCgamma1 microclusters. We conclude that HS1 functions as an effector of Itk in the T cell actin-regulatory pathway, and directs the spatial organization of PLCgamma1 signaling complexes.

Regulation of cell migration and morphogenesis by Abl-family kinases: emerging mechanisms and physiological contexts

The Abl-family non-receptor tyrosine kinases are essential regulators of the cytoskeleton. They transduce diverse extracellular cues into cytoskeletal rearrangements that have dramatic effects on cell motility and morphogenesis. Recent biochemical and genetic studies have revealed several mechanisms that Abl-family kinases use to mediate these effects. Abl-family kinases stimulate actin polymerization through the activation of cortactin, hematopoietic lineage cell-specific protein (HS1), WASp- and WAVE-family proteins, and Rac1. They also attenuate cell contractility by inhibiting RhoA and altering adhesion dynamics. These pathways impinge on several physiological processes, including development and maintenance of the nervous and immune systems, and epithelial morphogenesis. Elucidating how Abl-family kinases are regulated, and where and when they coordinate cytoskeletal changes, is essential for garnering a better understanding of these complex processes.

Hematopoietic lineage cell–specific protein 1 (HS1) is a functionally important signaling molecule in platelet activation

Collagen activates platelets through an intracellular signaling cascade downstream of glycoprotein VI (GPVI). We have investigated the contribution of hematopoietic lineage cell-specific protein 1 (HS1) downstream of GPVI in platelet activation. Stimulation of GPVI leads to tyrosine phosphorylation of HS1, which is blocked by Src-family kinase inhibitors. Coimmunoprecipitation experiments revealed that HS1 associates with Syk and phosphatidylinositol 3-kinases. HS1-null mice displayed increased bleeding times and increased time to occlusion in the FeCl(3) in vivo thrombosis model compared with their wild-type littermates. In addition, aggregation and secretion responses were diminished in HS1-null mouse platelets after stimulation of GPVI and protease-activated receptor 4 (PAR-4) agonists compared with wild-type littermate mouse platelets. Finally, Akt phosphorylation was diminished after GPVI or PAR-4 stimulation in platelets from HS1-null mice compared with their wild-type littermates. These results demonstrate that phosphorylation of the HS1 protein occurs downstream of GPVI stimulation and that HS1 plays a significant functional role in platelet activation downstream of GPVI and PARs.

The Coiled-coil Domain Is Required for HS1 to Bind to F-actin and Activate Arp2/3 Complex

HS1 (hematopoietic lineage cell-specific protein 1), a substrate of protein tyrosine kinases in lymphocytes, binds to F-actin, and promotes Arp2/3 complex-mediated actin polymerization. However, the mechanism for the interaction between HS1 and F-actin has not yet been fully characterized. HS1 contains 3.5 tandem repeats, a coiled-coil region, and an SH3 domain at the C terminus. Unlike cortactin, which is closely related to HS1 and requires absolutely the repeat domain for F-actin binding, an HS1 mutant with deletion of the repeat domain maintains a significant F-actin binding activity. On the other hand, deletion of the coiled-coil region abolished the ability of HS1 to bind to actin filaments and to activate the Arp2/3 complex for actin nucleation and actin branching. Furthermore, a peptide containing the coiled-coil sequence only was sufficient for F-actin binding. Within cells overexpressing green fluorescent protein-tagged HS1 proteins, wild type HS1 co-localizes with cortical F-actin at the cell leading edge, whereas mutants with deletion of either the coiled-coil region or the repeat domain diffuse in the cytoplasm. Immunoprecipitation analysis reveals that the coiled-coil deletion mutant binds poorly to F-actin, whereas the mutant without the repeat domain fails to bind to both Arp2/3 complex and F-actin. These data suggest that the HS1 coiled-coil region acts synergistically with the repeat domain in the modulation of the Arp2/3 complex-mediated actin polymerization.

Thrombin-induced Tyrosine Phosphorylation of HS1 in Human Platelets Is Sequentially Catalyzed by Syk and Lyn Tyrosine Kinases and Associated with the Cellular Migration of the Protein

Thrombin stimulation of platelets triggers Tyr phosphorylation of several signaling proteins, most of which remain unidentified. In this study, we demonstrate for the first time that hematopoietic lineage cell-specific protein 1 (HS1) undergoes a transient Tyr phosphorylation in human platelets stimulated with thrombin. The protein is synergistically phosphorylated by Syk and Lyn tyrosine kinases according to a sequential phosphorylation mechanism. By means of specific inhibitors (PP2, SU6656, and piceatannol) and phosphopeptide-specific antibodies, as well as by coimmunoprecipitation and binding competition experiments, we show that Syk acts as the primary kinase that phosphorylates HS1 at Tyr397 and that Syk phosphorylation is required for HS1 interaction with the Lyn SH2 domain. Upon docking to Syk-phosphorylated HS1, Lyn catalyzes the secondary phosphorylation of the protein at Tyr222. Once the secondary Tyr phosphorylation of HS1 is accomplished the protein dissociates from Lyn and undergoes a dephosphorylation process. HS1 Tyr phosphorylation does not occur when thrombin-induced actin assembly is inhibited by cytochalasin D even under conditions in which Syk and Lyn are still active. Immunofluorescence microscopic analysis shows that the agonist promotes HS1 migration to the plasma membrane and that the inhibition of Lyn-mediated secondary phosphorylation of HS1 abrogates the subcellular translocation of the protein. All together these results indicate that HS1 Tyr phosphorylation catalyzed by Syk and Lyn plays a crucial role in the translocation of the protein to the membrane and is involved in the cytoskeleton rearrangement triggered by thrombin in human platelets.

HS1 protein is differentially expressed in chronic lymphocytic leukemia patient subsets with good or poor prognoses

We used a proteomic approach for identifying molecules involved in the pathogenesis of chronic lymphocytic leukemia (CLL). We investigated 14 patients who were completely concordant for IgV(H) mutational status (unmutated vs. mutated), CD38 expression (positive vs. negative), and clinical behavior (progressive vs. stable); these patients were characterized as having either poor or good prognoses. The 2 patient subsets differed in the expression of hematopoietic lineage cell-specific protein 1 (HS1). In patients with poor prognoses, most HS1 protein was constitutively phosphorylated, whereas only a fraction was phosphorylated in patients with good prognoses. This difference was investigated in a larger cohort of 26 unselected patients. The survival curve of all 40 patients analyzed revealed that patients with predominately phosphorylated HS1 experience a significantly shorter median survival time. As HS1 is a protein pivotal in the signal cascade triggered by B cell receptor (BCR) stimulation, we studied its pattern of expression following BCR engagement. Normal mature B cells stimulated by anti-IgM shifted the non- or less-phosphorylated form of HS1 toward the more phosphorylated form. Naive B cells showed both HS1 forms while memory B cells expressed mainly the phosphorylated fraction. These data indicate a central role for antigen stimulation in CLL and suggest a new therapeutic target for patients with aggressive disease.

Syk-mediated Tyrosine Phosphorylation Is Required for the Association of Hematopoietic Lineage Cell-specific Protein 1 with Lipid Rafts and B Cell Antigen Receptor Signalosome Complex

Hematopoietic lineage cell-specific protein 1 (HS1) is an F-actin- and actin-related proteins 2 and 3 (Arp2/3)-binding protein that undergoes a rapid tyrosine phosphorylation upon B cell antigen receptor (BCR) activation. Density gradient centrifugation of Triton X-100 lysates from B lymphocytes demonstrated that HS1 was translocated in response to BCR cross-linking into lipid raft microdomain along with Arp2/3 complex and Wiskott-Aldrich syndrome protein. HS1-green fluorescent protein was localized in membrane patches enriched with GM1 gangliosides and BCR in the cells treated with anti-IgM antibody. Colocalization of HS1-green fluorescent protein with BCR was also correlated with tyrosine phosphorylation of HS1. Interestingly a murine HS1 mutant at the tyrosine residues Tyr388 and Tyr405 targeted by Syk failed to respond to BCR cross-linking for either translocation into lipid rafts or colocalization with BCR within cells. Furthermore HS1 was unable to translocate into lipid rafts in a chicken B cell line deficient in Syk. Reintroducing a Syk construct into the Syk knock-out cells recovered effectively both tyrosine phosphorylation and translocation of HS1 into lipid rafts. In contrast, translocation of HS1 into rafts was normal in a Lyn knock-out B cell line, and an HS1 mutant at the tyrosine residue Tyr222 targeted by Lyn maintained the ability to partition into rafts upon BCR cross-linking. These data indicate that Syk plays an important role in the translocation of HS1 into lipid rafts and may be responsible for actin assembly recruitment to rafts and subsequent antigen presentations.

Ser/Thr phosphorylation of hematopoietic specific protein 1 (HS1). Implication of protein kinase CK2

Hematopoietic lineage cell-specific protein 1 (HS1), a tyrosine multiphosphorylated protein implicated in receptor-mediated apoptosis and proliferative responses, is shown here to become Ser/Thr phosphorylated upon incubation of platelets with radiolabeled inorganic phosphate. The in vivo Ser/Thr phosphorylation of HS1 is enhanced by okadaic acid and reduced by specific inhibitors of casein kinase (CK)2. In vitro, HS1 is an excellent substrate for either CK2 α subunit alone (Km = 47 nm) or CK2 holoenzyme, tested in the presence of polylysine (Km = 400 nm). Phosphorylation reaches a stoichiometry of about 2 mol phosphate per mol HS1 and occurs mainly at threonyl residue(s), mostly located in the N-terminal region, but also at seryl residue(s) residing in the central core of the molecule (208–402), as judged from experiments with deleted forms of HS1. Ser/Thr phosphorylation of HS1, either induced in vivo by okadaic acid or catalysed in vitro by CK2, potentiates subsequent phosphorylation at tyrosyl residues. These data indicate the possibility that regulation of HS1 may also be under the control of Ser/Thr phosphorylation, and suggest that in quiescent cells CK2 could play a role in inducing constitutive Tyr phosphorylation of HS1 in the absence of stimuli that activate the protein tyrosine kinase pathway.

Ser/Thr phosphorylation of hematopoietic specific protein 1 (HS1): implication of protein kinase CK2.

Hematopoietic lineage cell-specific protein 1 (HS1), a tyrosine multiphosphorylated protein implicated in receptor-mediated apoptosis and proliferative responses, is shown here to become Ser/Thr phosphorylated upon incubation of platelets with radiolabeled inorganic phosphate. The in vivo Ser/Thr phosphorylation of HS1 is enhanced by okadaic acid and reduced by specific inhibitors of casein kinase (CK)2. In vitro, HS1 is an excellent substrate for either CK2 alpha subunit alone (Km = 47 nM) or CK2 holoenzyme, tested in the presence of polylysine (Km = 400 nM). Phosphorylation reaches a stoichiometry of about 2 mol phosphate per mol HS1 and occurs mainly at threonyl residue(s), mostly located in the N-terminal region, but also at seryl residue(s) residing in the central core of the molecule (208-402), as judged from experiments with deleted forms of HS1. Ser/Thr phosphorylation of HS1, either induced in vivo by okadaic acid or catalysed in vitro by CK2, potentiates subsequent phosphorylation at tyrosyl residues. These data indicate the possibility that regulation of HS1 may also be under the control of Ser/Thr phosphorylation, and suggest that in quiescent cells CK2 could play a role in inducing constitutive Tyr phosphorylation of HS1 in the absence of stimuli that activate the protein tyrosine kinase pathway.