Prolonged ERK Phosphorylation Research Articles

Investigating combination benefit of PD1 and LAG3 co-blockade using an engineered cellular bioassay.

LAG3 and PD1 are both immunomodulatory receptor that act by inhibiting activation of T cells, producing a more immunosuppressive environment. Even with the recent clinical success of PD1 and LAG3 co-blockade, signal transduction downstream of LAG3 remains largely unknown. We have leveraged an engineered Jurkat (T cell) and Raji (APC) co-culture system to assess simultaneous blockade of PD1 and LAG3 pathways using antibodies. RNA-Seq analysis of cell pellets individually treated with LAG3 or PD1 antibodies revealed modest immune activation however, 5-fold more genes were upregulated upon combination treatment. There were increases in costimulatory genes like CD28, CD5, CD6 as well as intracellular signaling molecules like LCP2 and ITK. Given the role of ERK in immune activation of T cells, pERK levels of Jurkat cells in the assay were evaluated. A very modest activation of pERK was observed with anti-LAG3 compared to anti-PD1 but a combination treatment resulted in prolonged ERK phosphorylation. Treatment of Jurkat cells with a commercial phosphatase inhibitor NSC87877 which can impact many phosphatases resulted in immune activation, measured by increased IL2 levels, only in the presence of LAG3. When NSC87877 was combined with the PD1 antibody, it could phenocopy combination benefit of PD1 and LAG3 blocking antibodies. CD28 has a recognized role in PD1 signaling but the impact on LAG3 signaling remains unknown. CD28 knockout in Jurkat cells affected overall IL-2 response of both LAG3 and PD1 antibody treatment but still retained combination benefit. Taken together this reductionist system highlights differences in downstream effects of LAG3 and PD1 blockade and we believe that the assay may have further utility to dissect convergence of both signaling pathways and augment studies in primary cells.

WWOX possesses N‐terminal cell surface‐exposed epitopes WWOX7‐21 and WWOX7‐11 for signaling cancer growth suppression and prevention in vivo

Membrane hyaluronidase Hyal‐2 supports cancer cell growth. Inhibition of Hyal‐2 by specific antibody against Hyal‐2 or pY216‐Hyal‐2 leads to cancer growth suppression and prevention in vivo. By immunoelectron microscopy, tumor suppressor WWOX is shown to be anchored, in part, in the cell membrane by Hyal‐2. Alternatively, WWOX undergoes self‐polymerization and localizes in the cell membrane. Proapoptotic pY33‐WWOX binds Hyal‐2, and TGF‐β induces internalization of the pY33‐WWOX/Hyal‐2 complex to the nucleus for causing cell death. In contrast, when pY33 is downregulated and pS14 upregulated in WWOX, pS14‐WWOX supports cancer growth in vivo. Here, we investigated whether membrane WWOX receives extracellular signals via surface‐exposed epitopes, especially at the S14 area, that signals for cancer growth suppression and prevention. By using a simulated 3‐dimentional structure and generated specific antibodies, WWOX epitopes were determined at amino acid #7 to 21 and #286 to 299. Synthetic WWOX7‐21 peptide, or truncation to 5‐amino‐acid WWOX7‐11, significantly suppressed and prevented the growth and metastasis of melanoma and skin cancer cells in mice. Time‐lapse microscopy revealed that WWOX7‐21 peptide potently enhanced the explosion and death of 4T1 breast cancer stem cell spheres by ceritinib. This is due to rapid upregulation of proapoptotic pY33‐WWOX, downregulation of prosurvival pERK, prompt increases in Ca+2 influx, and disruption of the IκBα/WWOX/ERK prosurvival signaling. In contrast, pS14‐WWOX7‐21 peptide dramatically increased cancer growth in vivo and protected cancer cells from ceritinib‐mediated apoptosis in vitro, due to a prolonged ERK phosphorylation. Further, specific antibody against pS14‐WWOX significantly enhanced the ceritinib‐induced apoptosis. Together, the N‐terminal epitopes WWOX7‐21 and WWOX7‐11 are potent in blocking cancer growth in vivo. WWOX7‐21 and WWOX7‐11 peptides and pS14‐WWOX antibody are of therapeutic values in suppressing and preventing cancer growth in vivo.Support or Funding InformationMinistry of Science and Technology, Taiwan, National Health Research Institute, Taiwan, and Department of Defense, USAWWOX peptides regulate breast cancer cell sensitivity to ceritinib.Figure 1

WWOX Possesses N-Terminal Cell Surface-Exposed Epitopes WWOX7-21 and WWOX7-11 for Signaling Cancer Growth Suppression and Prevention In Vivo.

Membrane hyaluronidase Hyal-2 supports cancer cell growth. Inhibition of Hyal-2 by specific antibody against Hyal-2 or pY216-Hyal-2 leads to cancer growth suppression and prevention in vivo. By immunoelectron microscopy, tumor suppressor WWOX is shown to be anchored, in part, in the cell membrane by Hyal-2. Alternatively, WWOX undergoes self-polymerization and localizes in the cell membrane. Proapoptotic pY33-WWOX binds Hyal-2, and TGF-β induces internalization of the pY33-WWOX/Hyal-2 complex to the nucleus for causing cell death. In contrast, when pY33 is downregulated and pS14 upregulated in WWOX, pS14-WWOX supports cancer growth in vivo. Here, we investigated whether membrane WWOX receives extracellular signals via surface-exposed epitopes, especially at the S14 area, that signals for cancer growth suppression and prevention. By using a simulated 3-dimentional structure and generated specific antibodies, WWOX epitopes were determined at amino acid #7 to 21 and #286 to 299. Synthetic WWOX7-21 peptide, or truncation to 5-amino acid WWOX7-11, significantly suppressed and prevented the growth and metastasis of melanoma and skin cancer cells in mice. Time-lapse microscopy revealed that WWOX7-21 peptide potently enhanced the explosion and death of 4T1 breast cancer stem cell spheres by ceritinib. This is due to rapid upregulation of proapoptotic pY33-WWOX, downregulation of prosurvival pERK, prompt increases in Ca2+ influx, and disruption of the IkBα/WWOX/ERK prosurvival signaling. In contrast, pS14-WWOX7-21 peptide dramatically increased cancer growth in vivo and protected cancer cells from ceritinib-mediated apoptosis in vitro, due to a prolonged ERK phosphorylation. Further, specific antibody against pS14-WWOX significantly enhanced the ceritinib-induced apoptosis. Together, the N-terminal epitopes WWOX7-21 and WWOX7-11 are potent in blocking cancer growth in vivo. WWOX7-21 and WWOX7-11 peptides and pS14-WWOX antibody are of therapeutic values in suppressing and preventing cancer growth in vivo.

5α-Androstane-3α,17β-Diol Inhibits Neurotoxicity in SH-SY5Y Human Neuroblastoma Cells and Mouse Primary Cortical Neurons.

Low free T levels in men are associated with age-related cognitive decline and increased risk for neurotoxicity, resulting in disease. The mechanisms underlying these observations remain poorly defined. Although rapid, androgen receptor-dependent activation of ERK has been postulated as a neurotrophic and neuroprotective mechanism, actions of T metabolites such as 5α-androstane-3α,17β-diol (3α-diol) may also be involved. We investigated the influence of 3α-diol on the induction of ERK phosphorylation in SH-SY5Y human female neuroblastoma cells and primary cortical neurons from male and female mice. In SH-SY5Y cells, ERK phosphorylation was induced by 10 nM DHT, epidermal growth factor, hydrogen peroxide (H2O2), and acetylcholine. The addition of 10 nM 3α-diol, which did not itself activate ERK, significantly inhibited ERK phosphorylation induced by DHT, epidermal growth factor, or H2O2, but not acetylcholine. In both SH-SY5Y cells and primary cortical neurons, prolonged ERK phosphorylation and caspase-3 cleavage resulting from amyloid β-peptide 1-42 (Aβ42) exposure were inhibited by cotreatment with 3α-diol. 3α-diol also reduced the loss in cellular viability induced by Aβ42 or H2O2 in SH-SY5Y cells. These data suggest that T-mediated neuroprotection may occur via two distinct but complementary mechanisms: an initial rapid activation of ERK phosphorylation, followed by modulation via 3α-diol of the potentially adverse consequences of prolonged ERK activation.

Hypoxia-Induced MicroRNA-20a Expression Increases ERK Phosphorylation and Angiogenic Gene Expression in Endometriotic Stromal Cells

Aberrant activation of MAPK has been implicated to play important roles in pathological processes of endometriosis. However, how MAPK are constitutively activated in endometriotic tissues remains largely unknown. microRNA are small noncoding RNA that regulate the stability or translational efficiency of target mRNA by interacting with the 3' untranslated region. Thus, miRNA are thought to be modulators of the transcriptional response, fine-tuning gene expression. The aim of this study was to evaluate the functional roles of microRNA-20a (miR20a) in MAPK activation and the pathogenesis of endometriosis. miR20a expression was analyzed in nonpaired (endometrium = 17; endometriosis = 37) and paired (n = 12) endometriotic tissues by quantitative RT-PCR. Overexpression of miR20a in eutopic endometrial stromal cells or inhibition of miR20a in ectopic endometriotic stromal cells was used to evaluate its impact on ERK phosphorylation and subsequently angiogenesis- and proliferation-related gene expression. Levels of miR20a were up-regulated in endometriotic stromal cells. Elevation of miR20a was up-regulated by hypoxia inducible factor-1α. The up-regulation of miR20a causes the down-regulation of dual-specificity phosphatase-2, which leads to prolonged ERK phosphorylation and an increase in the expression of several angiogenic genes. Furthermore, the up-regulation of miR20a enhances the prostaglandin E(2)-induced expression of fibroblast growth factor-9, a potent mitogen that stimulates both endothelial and endometrial cell proliferation. Our findings provide the novel mechanism that not only functionally links together hypoxic stress, miR20a expression, aberrant ERK phosphorylation, and angiogenesis but also demonstrates that miR20a is an important modulator in the development of endometriosis.

Hyaluronan suppresses prostate tumor cell proliferation through diminished expression of N-cadherin and aberrant growth factor receptor signaling

Hyaluronan (HA) production has been functionally implicated in prostate tumorigenesis and metastasis. We previously used prostate tumor cells overexpressing the HA synthesizing enzyme HAS3 or the clinically relevant hyaluronidase Hyal1 to show that excess HA production suppresses tumor growth, while HA turnover accelerates spontaneous metastasis from the prostate. Here, we examined pathways responsible for effects of HAS3 and Hyal1 on tumor cell phenotype. Detailed characterization of cell cycle progression revealed that expression of Hyal1 accelerated cell cycle re-entry following synchronization, whereas HAS3 alone delayed entry. Hyal1 expressing cells exhibited a significant reduction in their ability to sustain ERK phosphorylation upon stimulation by growth factors, and in their expression of the cyclin-dependent kinase inhibitor p21. In contrast, HAS3 expressing cells showed prolonged ERK phosphorylation and increased expression of both p21 and p27, in asynchronous and synchronized cultures. Changes in cell cycle regulatory proteins were accompanied by HA-induced suppression of N-cadherin, while E-cadherin expression and β-catenin expression and distribution remained unchanged. Our results are consistent with a model in which excess HA synthesis suppresses cell proliferation by promoting homotypic E-cadherin mediated cell–cell adhesion, consequently signaling to elevate cell cycle inhibitor expression and suppress G1- to S-phase transition.

Estrogen Receptor-α But Not -β or GPER Inhibits High Glucose-Induced Human VSMC Proliferation: Potential Role of ROS and ERK

The decreased incidence of cardiovascular disease in premenopausal women has been attributed, at least partially, to protective effects of estrogens. However, premenopausal women with diabetes mellitus are no longer selectively protected. High-glucose (HG) conditions have previously been shown to abolish the antimitogenic effects of 17β-estradiol (E(2)) in vascular smooth muscle cells (VSMCs). Because E(2) mediates its action via different estrogen receptor (ER) subtypes, we hypothesized that different subtypes may have different, if not opposing, effects on HG-induced VSMC proliferation. Treatment of human aortic VSMCs isolated from premenopausal women with the selective ERα agonist, 4,4',4'-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol, but not with E(2), the selective ERβ agonist 2,3-bis(4-hydroxyphenyl)-propionitrile, or the selective G protein-coupled ER agonist G-1 completely prevented increased HG-induced VSMC proliferation. Under these conditions, ERα activation selectively prevented increased hydrogen peroxide (H(2)O(2)) and total intracellular reactive oxygen species (ROS) production, caused up-regulation of manganese superoxide dismutase protein and activity, and inhibited prolonged ERK phosphorylation. The latter was mediated by ROS, and ROS inhibition reversed HG-induced ERK-dependent VSMC proliferation. The selective coactivation of ERβ reversed the antimitogenic and antioxidative effects of ERα as well as the up-regulation of manganese superoxide dismutase protein expression. Selective activation of ERα is required for reducing oxidative stress and the consequent hyperproliferation of VSMCs under HG. Our results may further suggest that ERα activation inhibits HG-induced proliferation by down-regulating ROS-mediated ERK activation and may explain why antimitogenic effects of E(2) are abolished under HG. Pharmacological activation of ERα may thus have therapeutic potential for treating cardiovascular dysregulation associated with diabetes.

BLNK Binds Active H-Ras to Promote B Cell Receptor-mediated Capping and ERK Activation

Cross-linked B cell receptor (BCR) aggregates on the cell surface, then assembles into the "cap" where Ras is co-localized, and transduces various intracellular signals including Ras-ERK activation. BCR signals induce proliferation, differentiation, or apoptosis of B cells depending on their maturational stage. The adaptor protein BLNK binds various signaling proteins and Igalpha, a signaling subunit of the BCR complex, and plays an important role in the BCR signal transduction. BLNK was shown to be required for activation of ERK, but not of Ras, after BCR cross-linking, raising a question how BLNK facilitates ERK activation. Here we demonstrate that BLNK binds the active form of H-Ras, and their binding is facilitated by BCR cross-linking. We have identified a 10-amino acid Ras-binding domain within BLNK that is necessary for restoration of BCR-mediated ERK activation in BLNK-deficient B cells and for anti-apoptotic signaling. The Ras-binding domain fused with a CD8alpha-Igalpha chimeric receptor could induce prolonged ERK phosphorylation, transcriptional activation of Elk1, as well as the capping of the receptor in BLNK-deficient B cells. These results indicate that BLNK recruits active H-Ras to the BCR complex, which is essential for sustained surface expression of BCR in the form of the cap and for the signal leading to functional ERK activation.

Changes in apical dendritic structure correlate with sustained ERK1/2 phosphorylation in medial prefrontal cortex of a rat model of dopamine D1 receptor agonist sensitization.

Rats lesioned with 6-hydroxydopamine (6-OHDA) as neonates exhibit behavioral and neurochemical abnormalities in adulthood that mimic Lesch-Nyhan disease, schizophrenia, and other developmental disorders of frontostriatal circuit dysfunction. In these animals a latent sensitivity to D1 agonists is maximally exposed by repeated administration of dopamine agonists in the postpubertal period (D1 priming). In neonate-lesioned, adult rats primed with SKF-38393, we found selective, persistent alterations in the morphology of pyramidal neuron apical dendrites in the prelimbic area of the medial prefrontal cortex (mPFC). In these animals, dendrite bundling patterns and the typically straight trajectories of primary dendritic shafts were disrupted, whereas the diameter of higher-order oblique branches was increased. Although not present in neonate-lesioned rats treated with saline, these morphological changes persisted at least 21 days after repeated dosing with SKF-38393, and were not accompanied by markers of neurodegenerative change. A sustained increase in phospho-ERK immunoreactivity in wavy dendritic shafts over the same period suggested a relationship between prolonged ERK phosphorylation and dendritic remodeling in D1-primed rats. In support of this hypothesis, pretreatment with the MEK1/2-ERK1/2 pathway inhibitors PD98059 or SL327, prior to each priming dose of SKF-38393, prevented the morphological changes associated with D1 priming. Together, these findings demonstrate that repeated stimulation of D1 receptors in adulthood interacts with the developmental loss of dopamine to profoundly and persistently modify neuronal signaling and dendrite morphology in the mature prefrontal cortex. Furthermore, sustained elevation of ERK activity in mPFC pyramidal neurons may play a role in guiding these morphological changes in vivo.

IgE-Induced Mast Cell Survival Requires the Prolonged Generation of Reactive Oxygen Species

We show in this study that the ability of five different monomeric IgEs to enhance murine bone marrow-derived mast cell (BMMC) survival correlates with their ability to stimulate extracellular calcium (Ca(2+)) entry. However, whereas IgE+Ag more potently stimulates Ca(2+) entry, it does not enhance survival under our conditions. Exploring this further, we found that whereas all five monomeric IgEs stimulate a less robust Ca(2+) entry than IgE+Ag initially, they all trigger a more prolonged Ca(2+) influx, generation of reactive oxygen species (ROS), and ERK phosphorylation. These prolonged signaling events correlate with their survival-enhancing ability and positively feedback on each other to generate the prosurvival cytokine, IL-3. Interestingly, the prolonged ERK phosphorylation induced by IgE appears to be regulated by a MAPK phosphatase rather than MEK. IgE-induced ROS generation, unlike that triggered by IgE+Ag, is not mediated by 5-lipoxygenase. Moreover, ROS inhibitors, which block both IgE-induced ROS production and Ca(2+) influx, convert the prolonged ERK phosphorylation induced by IgE into the abbreviated phosphorylation pattern observed with IgE+Ag and prevent IL-3 generation. In support of the essential role that IgE-induced ROS plays in IgE-enhanced BMMC survival, we found the addition of H(2)O(2) to IgE+Ag-stimulated BMMCs leads to IL-3 secretion.

Genetic Disruption of Rac1 but Not Rac2 Is Sufficient To Ameliorate the Development of Juvenile Myeloproliferative Myeloid Leukemia in Nf1−/− Mice.

Neurofibromin, the protein encoded by the NF1 tumor-suppressor gene, negatively regulates the output of p21ras proteins by accelerating the hydrolysis of active Ras-guanosine triphosphate to inactive Ras-guanosine diphosphate. Children with neurofibromatosis type 1 (NF1) are predisposed to juvenile myelomonocytic leukemia (JMML) associated with loss of the normal NF1 allele. Genetically engineered mice containing nullizygous loss of Nf1 in the hematopoietic system develop a leukemia with complete penetrance that is similar to JMML, including hepatosplenomegaly, elevated peripheral blood cell counts, and elevated numbers of myeloid progenitors that are hypersensitive to multiple cytokines, particularly GM-CSF. Though we and others have found that p21ras is hyperactivated in Nf1−/− myeloid progenitors, inhibiting Ras specifically as a therapeutic target has been challenging. Therefore, identification of alterations in distinct p21ras effector pathways that control leukemia progression in Nf1-deficient cells is critical for understanding disease pathogenesis and identifying therapeutic targets. Here we intercrossed MxCre; Nf1flox/flox mice with mice that are deficient in the small Rho GTPases Rac1 or Rac2 to generate syngeneic progeny that were MxCre; Nf1flox/flox, MxCre; Nf1flox/flox;Rac1flox flox or MxCre; Nf1flox/flox; Rac2 −/−. Consistent with previous studies (Le, Blood 2004) MxCre; Nf1flox/flox mice develop a progressive myeloproliferative disease with 100% penetrance 6 months following interferon inducible induction of the MxCre transgene to disrupt the Nf1flox alleles. Eighty percent of MxCre; Nf1flox/flox die by 9 months after inactivation. Genetic disruption of Rac2 was not sufficient to diminish the onset or severity of the characteristic myeloproliferative disease of MxCre; Nf1flox/flox mice. In contrast, MxCre; Nf1flox/flox; Rac1flox/flox mice followed for 11 months all survived and had normal bone marrow cellularity, spleen weight and splenic architecture. MxCre; Nf1flox/flox mice have elevated numbers of both HPP-CFC and LPP-CFC in the bone marrow and spleen. In contrast, MxCre; Nf1flox/flox; Rac1flox/flox mice had myeloid progenitor numbers that were comparable to wildtype, age-matched controls. Further, though myeloid progenitors from MxCre; Nf1flox/flox mice are hypersensitive to GM-CSF, myeloid progenitors from MxCre; Nf1flox/flox; Rac1flox/flox have a sensitivity to GM-CSF comparable to wildtype controls. The correction in the myeloproliferative phenotype isolated from MxCre; Nf1flox/flox; Rac1flox/flox mice was associated with the reduction in Rac-GTP and prolonged Erk phosphorylation, a MAPK effector that is characteristically elevated in Nf1−/− myeloid progenitors. Collectively, these genetic data identify Ras-Rac1 signaling pathway as a key axis in the genesis of juvenile myelomonocytic leukemia and provide evidence that Rac1 is a therapeutic molecular target for this myeloproliferative disease that currently has no effective therapies.

Phosphorylation regulates Myc expression via prolonged activation of the mitogen‐activated protein kinase pathway

We previously showed that prolonged and strong ERK phosphorylation induced by Compound 5 (Cpd 5), a Cdc25A protein phosphatase inhibitor, was involved in its mechanism of cell growth inhibition. To study the relationship between ERK phosphorylation and cell growth inhibition, we used Cpd 5 as a tool to investigate ERK-regulated c-Myc expression in Hep3B hepatoma cells. We found that ERK phosphorylation caused by Cpd 5 induced c-Myc phosphorylation, but suppressed c-Myc expression at the mRNA and protein levels. Furthermore, Cpd 5 inhibited c-Myc transcriptional activity and DNA binding ability, and this inhibition was antagonized by ERK kinase (MEK) inhibitor U-0126, implying that the ERK pathway was involved in regulating c-Myc expression. Since the participation of c-Myc protein in transcription requires its dimerization with Max protein, we examined the Myc-Max association in Cpd 5-treated cells and found that Cpd 5 suppressed Myc-Max dimerization. Transfection of Hep3B cells with mutated ERK (T188A/Y190F), which has lost its dual-phosphorylation sites, attenuated the actions of Cpd 5 on Myc-Max association. To further demonstrate whether Myc phosphorylation by Cpd 5-induced ERK activation was able to directly regulate c-myc gene expression, a chromatin immunoprecipitation (ChIP) assay was used to examine the binding of phospho-Myc to the c-myc promoter region. We found that phospho-Myc induced by Cpd 5 had lost its ability to bind to the c-myc promoter, whereas MEK inhibitor U-0126 antagonized this inhibitory effect. These data suggest that an increase in c-Myc phosphorylation in response to prolonged ERK phosphorylation negatively auto-regulates c-Myc gene expression, leading to the suppression of its target gene expression and cell cycle block.

CXCL-12 Induces Prolonged Erk-Phosphorylation in Zap-70 Positive B-CLL.

IntroductionZap70 is superior to mutation status as a prognostic indicator in B-CLL, but whether it is causally implicated or just associated with progressive disease is unclear. Zap-70 transduces the TCR signal through the CD3 zeta-unit in T cells, but although it is important in preB development, it has no known function in mature B-cells. Zap-70 positive B-CLL show enhanced B cell receptor mediated Syk-phosphorylation upon surface IgM (sIgM) crosslinking, simulating antigen driven B cell proliferation. This may increase the proliferative drive in Zap-70+ B-CLL compared to Zap-70- leading to a higher risk of secondary chromosomal damage. Zap-70 was recently implied in Erk activation via CXCL-12/CXCR4 in T-cells and this is enhanced by cell adhesion through beta-1 integrins that may influence P-Erk phosphatases. Since both CXCL12/CXCR-4 and integrins influence B-CLL survival in vitro, we investigated the duration of downstream Erk activation in Zap-70 + versus Zap-70 B-CLL after CXCL-12 stimulation in the presence and absence of integrin binding by fibronectin.Materials/methodsPeripheral blood mononuclear cells from B-CLL patients were isolated by Ficoll-gradient. Zap-70 was measured by flow cytometry (Upstate Ab), and PCR.B-CLL cells from 6 patients were stimulated with 100 ng/ml CXCL-12 after preincubation in serum free RPMI for 2 hrs at 37°in fibronectin or BSA coated plates and in suspension culture. After CXCL-12 addition supernatant and adherent cells were separately lysed at 0, 2 and 10 minutes and studied by Western blot for total Erk, Akt, P-Erk and P-Akt. Parallel cultures were extended for 24 hours and apoptosis of supernatant versus adherent cells was performed by AnnexinV-PI staining and flow cytometry.ResultsErk-phosphorylation was markedly stimulated by CXCL-12 after 2 minutes, both in supernatant and adherent fractions of fibronectin and BSA coated plates compared to suspension cultures. This stimulation decreased in Zap-70- B-CLL after 10 minutes but remained high in Zap-70+ B-CLL particularly in the fibronectin adherent fraction. These cells were also protected from spontaneous apoptosis at 24 hours when compared to supernatant Zap70+ and Zap-70- cells.ConclusionCXCL-12/CXCR4 ligation leads to prolonged Erk-phosphorylation in Zap-70+ but not in Zap70- B-CLL cells. Prolonged Erk-activation is required for nuclear translocation and effective signal transduction along this way. This effect is enhanced by concomitant fibronectin engagement of integrins, which may give Zap-70+ B-CLL cells a proliferative advantage upon homing in lymphoid tissues.