Correction: Alcalá, S., et al. Targeting SRC Kinase Signaling in Pancreatic Cancer Stem Cells. Int. J. Mol. Sci. 2020, 21, 7437.

The authors recently reported on the potential of targeting SRC kinase signaling in pancreatic cancer stem cells [...].

Targeting SRC Kinase Signaling in Pancreatic Cancer Stem Cells.

The proto-oncogene nonreceptor tyrosine-protein kinase SRC is a member of the SRC family of tyrosine kinases (SFKs), and its activation and overexpression have been shown to play a protumorigenic role in multiple solid cancers, including pancreatic ductal adenocarcinoma (PDAC). PDAC is currently the seventh-leading cause of cancer-related death worldwide, and, by 2030, it is predicted to become the second-leading cause of cancer-related death in the United States. PDAC is characterized by its high lethality (5-year survival of rate of <10%), invasiveness, and chemoresistance, all of which have been shown to be due to the presence of pancreatic cancer stem cells (PaCSCs) within the tumor. Due to the demonstrated overexpression of SRC in PDAC, we set out to determine if SRC kinases are important for PaCSC biology using pharmacological inhibitors of SRC kinases (dasatinib or PP2). Treatment of primary PDAC cultures established from patient-derived xenografts with dasatinib or PP2 reduced the clonogenic, self-renewal, and tumor-initiating capacity of PaCSCs, which we attribute to the downregulation of key signaling factors such as p-FAK, p-ERK1-2, and p-AKT. Therefore, this study not only validates that SRC kinases are relevant and biologically important for PaCSCs but also suggests that inhibitors of SRC kinases may represent a possible future treatment option for PDAC patients, although further studies are still needed.

Role of SRC family kinases in prolactin signaling.

Prolactin (PRL) is a polypeptide hormone/cytokine mainly synthesized by the lactotrophic cells of the adenohypophysis. In addition to the best-known role in mammary gland development and the functional differentiation of its epithelium, PRL is involved in regulation of multiple physiological processes in higher organisms contributing to their homeostasis. PRL has been also associated with pathology, including breast cancer. Therefore, it is relevant to determine the molecular mechanisms by which PRL controls cellular functions. Here, we analyze the role of Src family kinases (SFKs) in the intracellular signaling pathways controlled by PRL in several model systems. The data show that SFKs are essential components in transmitting signals upon PRL receptor stimulation, as they control activation of Jak2/Stat5 and other routes that regulate PRL cellular responses.

Protein tyrosine phosphatases as novel targets in breast cancer therapy.

Breast cancer is linked to hyperactivation of protein tyrosine kinases (PTKs), and recent studies have unveiled that selective tyrosine dephosphorylation by protein tyrosine phosphatases (PTPs) of specific substrates, including PTKs, may activate or inactivate oncogenic pathways in human breast cancer cell growth-related processes. Here, we review the current knowledge on the involvement of PTPs in breast cancer, as major regulators of breast cancer therapy-targeted PTKs, such as HER1/EGFR, HER2/Neu, and Src. The functional interplay between PTKs and PTK-activating or -inactivating PTPs, and its implications in novel breast cancer therapies based on targeting of specific PTPs, are discussed.

The Relevance of the SH2 Domain for c-Src Functionality in Triple-Negative Breast Cancer Cells.

The role of Src family kinases (SFKs) in human tumors has been always associated with tyrosine kinase activity and much less attention has been given to the SH2 and SH3 adapter domains. Here, we studied the role of the c-Src-SH2 domain in triple-negative breast cancer (TNBC). To this end, SUM159PT and MDA-MB-231 human cell lines were employed as model systems. These cells conditionally expressed, under tetracycline control (Tet-On system), a c-Src variant with point-inactivating mutation of the SH2 adapter domain (R175L). The expression of this mutant reduced the self-renewal capability of the enriched population of breast cancer stem cells (BCSCs), demonstrating the importance of the SH2 adapter domain of c-Src in the mammary gland carcinogenesis. In addition, the analysis of anchorage-independent growth, proliferation, migration, and invasiveness, all processes associated with tumorigenesis, showed that the SH2 domain of c-Src plays a very relevant role in their regulation. Furthermore, the transfection of two different aptamers directed to SH2-c-Src in both SUM159PT and MDA-MB-231 cells induced inhibition of their proliferation, migration, and invasiveness, strengthening the hypothesis that this domain is highly involved in TNBC tumorigenesis. Therefore, the SH2 domain of c-Src could be a promising therapeutic target and combined treatments with inhibitors of c-Src kinase enzymatic activity may represent a new therapeutic strategy for patients with TNBC, whose prognosis is currently very negative.

HCT116 cells deficient in p21(Waf1) are hypersensitive to tyrosine kinase inhibitors and adriamycin through a mechanism unrelated to p21 and dependent on p53.

p21(Waf1) (p21) was described as a cyclin-dependent kinase inhibitor, but other p21 activities have subsequently been described, including its ability to inhibit apoptosis in some models. Comparative work on the human colon cancer isogenic cell lines HCT116 and HCT116p21(-/-) led to the proposal that p21 protects colon cancer cells against apoptosis by genotoxic drugs. We asked whether p21 also protected from cell death induced by non-genotoxic drugs, such as tyrosine kinase inhibitors. We found that p21-deficient cells were dramatically more sensitive towards imatinib and gefitinib than parental cells. Interestingly, HCT116p21(-/-) also showed higher basal activity of protein kinases as c-Abl, c-Src, and Akt. We generated HCT116p21(-/-) sublines with inducible p21 expression and found that p21 did not rescue the hypersensitivity to imatinib. Moreover, down-regulation of p21 by enforced c-Myc expression or by p21 siRNA did not sensitize parental HCT116 cells. We found that, in HCT116p21(-/-) cells, p53 showed higher stability, higher transcriptional activity and phosphorylation in serines associated with p53 activity. Furthermore, silencing of p53 with siRNA and inactivation of p53 with a dominant negative mutant rescued the hypersensitive response to kinases inhibitors, 5-fluorouracil and adriamycin in HCT116p21(-/-) cells. Consistently, HCT116p53(-/-) cells are more resistant to imatinib than parental cells, suggesting that imatinib activity is partly dependent on p53 in colon cancer cells. We conclude that high p53 activity, rather than p21 deficiency, is the mechanism responsible for hypersensitivity to drugs of HCT116p21(-/-) cells. Therefore the role of p21 on apoptosis of HCT116 colon cancer cells should be re-evaluated.

c-Src functionality controls self-renewal and glucose metabolism in MCF7 breast cancer stem cells.

Deregulation of Src kinases is associated with cancer. We previously showed that SrcDN conditional expression in MCF7 cells reduces tumorigenesis and causes tumor regression in mice. However, it remained unclear whether SrcDN affected breast cancer stem cell functionality or it reduced tumor mass. Here, we address this question by isolating an enriched population of Breast Cancer Stem Cells (BCSCs) from MCF7 cells with inducible expression of SrcDN. Induction of SrcDN inhibited self-renewal, and stem-cell marker expression (Nanog, Oct3-4, ALDH1, CD44). Quantitative proteomic analyses of mammospheres from MCF7-Tet-On-SrcDN cells (data are available via ProteomeXchange with identifier PXD017789, project DOI: 10.6019/PXD017789) and subsequent GSEA showed that SrcDN expression inhibited glycolysis. Indeed, induction of SrcDN inhibited expression and activity of hexokinase, pyruvate kinase and lactate dehydrogenase, resulting in diminished glucose consumption and lactate production, which restricted Warburg effect. Thus, c-Src functionality is important for breast cancer stem cell maintenance and renewal, and stem cell transcription factor expression, effects linked to glucose metabolism reduction.

miR205 inhibits stem cell renewal in SUM159PT breast cancer cells.

miR205 has a dual activity, as tumor suppressor and as oncogene. Here we analyzed the impact of miR205 ectopic expression in the initial tumorigenic processes of SUM159PT, a triple negative breast cancer cell line with low endogenous levels of miR205. In SUM159PT, miR205 inhibited expression of its targets VEGFA, ErbB3, Zeb1, Fyn and Lyn A/B; it reduced cell proliferation, and Myc/cyclin D1 levels, while increased p27kip1 expression. miR205 abolished anchorage-independent growth, inhibited migration and invasion, Src-kinases/Stat3 axis activation, and levels of secreted MMP9. miR205 also reduced expression of CD44 and TAZ, E2A.E12, Twist, Snail1 and CK5, associated with epithelial-mesenchymal transition (EMT). Importantly, we show that miR205 inhibited SUM159PT cancer-stem cell renewal, expression in mammospheres of CD44 and ALDH1 stem-cell markers, TAZ, and E2A.E12. All these effects of miR205 were reverted by Anti-miR205 co-expression, demonstrating its specificity. Thus, all these results strongly suggest that ectopic expression of miR205 in SUM159PT affected several parameters associated with initial steps of tumorigenesis.

Reduced expression of the murine HLA-G homolog Qa-2 is associated with malignancy, epithelial-mesenchymal transition and stemness in breast cancer cells.

Qa-2 is believed to mediate a protective immune response against cancer; however, little is known about the role of Qa-2 in tumorigenesis. Here, we used 4T1 breast cancer cells to study the involvement of Qa-2 in tumor progression in a syngeneic host. Qa-2 expression was reduced during in vivo tumor growth and in cell lines derived from 4T1-induced tumors. Tumor-derived cells elicited an epithelial-mesenchymal transition associated with upregulation of Zeb1 and Twist1/2 and enhanced tumor initiating and invasive capacities. Furthermore, these cells showed increased stem characteristics, as demonstrated by upregulation of Hes1, Sox2 and Oct3/4, and enrichment of CD44high/CD24median/low cells. Remarkably, Qa-2 cell-surface expression was excluded from the CD44high/CD24median/low subpopulation. Tumor-derived cells showed increased Src activity, and treatment of these cells with the Src kinase inhibitor PP2 enhanced Qa-2 but reduced Sox2 and CD44high/CD24median/low expression levels, suggesting that Src signaling, while positively associated with stemness, negatively regulates Qa-2 expression in breast cancer. Finally, overexpression of the Qa-2 family member Q7 on the cell surface slowed down in vivo tumor growth and reduced the metastatic potential of 4T1 cells. These results suggest an anti-malignant role for Qa-2 in breast cancer development, which appears to be absent from cancer stem cells.

Cytoplasmic localization of p120ctn and E-cadherin loss characterize lobular breast carcinoma from preinvasive to metastatic lesions.

Accumulating evidences indicate that p120 catenin, a member of the E-cadherin (E-CD)/catenin adhesion complex, plays a role in tumor invasion. To establish the expression pattern of p120 in breast cancer, we analysed 326 breast tissue biopsies by tissue microarray. Most of the lobular tumors (88%) showed exclusive cytoplasmic localization, and 6% of them also had p120 nuclear staining. Cytoplasmic p120 strongly associated with complete loss of E-CD and beta-catenin not only in lobular carcinoma and its metastases but also in atypical lobular hyperplasias. In the latter, loss of heterozygosity of E-CD gene was also observed. Complete loss of E-CD and cytoplasmic and nuclear p120 staining was also observed in primary lobular cancer cell cultures generated by us. In ductal tumors, by contrast, reduction of p120 and E-CD in membrane was very common (57 and 53%, respectively), whereas cytoplasmic p120 staining was rarely seen. This simultaneous reduction of membranous E-CD and p120 was not associated with increased Src kinase activity. To demonstrate that cytoplasmic p120 localization was a consequence of the absence of E-CD, the endogenous E-CD was re-expressed in MDA-231 cells by 5-Aza-2'-deoxycytidine (5Aza) treatment. After treatment, p120 shifted from the cytoplasm to the membrane, where it colocalized with endogenous E-CD. Additionally, suppressing E-CD expression in Madin-Darby canine kidney cells by stable transfection of the transcriptional repressors Snail, E47 or Slug, provokes p120 cytoplasmic localization and p120 isoform switching. In conclusion, abnormal cytoplasmic and nuclear localization of p120, which are mediated by the absence of E-CD, characteristically occur in the early stages of lobular breast cancer and are maintained during tumor progression to metastasis. Consequently, p120 may be an important mediator of the oncogenic effects derived from E-CD inactivation, including enhanced motility and invasion, in lobular breast cancer.

Prolactin induces c-Myc expression and cell survival through activation of Src/Akt pathway in lymphoid cells.

Stimulation of resting W53 cells (lymphoid murine cells expressing prolactin (PRL) receptor) by PRL induced expression of growth-related immediate-early genes (IEG), and proliferation through activation of the Src kinases. Since IEG are essential for cell cycle progression, we have studied how PRL controls expression of c-Myc mRNA and c-Fos. Stimulation of W53 cell proliferation by PRL required activation of MAPK, as the Mek1/2 inhibitor PD184352 eliminated Erk1/2 stimulation, cell proliferation, and expression of c-Fos mRNA. In contrast, PD184352 did not alter PRL activation of c-Myc mRNA expression or stimulation of p70S6K, Akt, and the Jak2/Stat5 pathway. Activation of the PI3K by PRL was necessary for the expression of c-MycmRNA and W53 cell proliferation, as the PI3K inhibitor LY294002 abolished them. However, it did not modify PRL stimulation of c-Fos mRNA expression or activation of Erk1/2 and Stat5. Furthermore, rapamycin, an inhibitor of mTOR and consequently of p70S6K, did not alter PRL stimulation of c-Myc and c-Fos mRNA expression and it had a very minor inhibitory effect on PRL stimulation of W53 cell proliferation. In addition, rapamycin did not affect PRL stimulation of Akt or Stat5. However, it reinforced PRL activation of Erk1/2. Overexpression of a constitutively activated Akt (myristoylated Akt) in W53 cells overcame the inhibitory effect of LY294002 on c-Myc expression, as well as cell death upon PRL deprivation. Consistently, inducible expression of Akt-CAAX Box in W53 cells caused inhibition of c-Myc expression. PRL stimulation of W53 cells resulted in Akt translocation to the nucleus, phosphorylation of FKHRL1 transcription factor, and its nuclear exclusion. In contrast, induced expression of Akt-CAAX Box caused inhibition of FKHRL1 phosphorylation. Furthermore, transient expression of nonphosphorylatable FKHRL1-A3 mutant impaired PRL-induced activation of the c-Myc promoter. Akt activation also resulted in phosphorylation and inhibition of glycogen synthetase kinase 3 (GSK3), which in turn promoted c-Myc stability. Consistently, treatment of W53 with selective inhibitors of GSK3 such as SB415286 and lithium salts resulted in increased levels of c-Myc. Also, overexpression of c-Myc in W53 cells overcame the decrease in cell proliferation induced by LY294002. These findings defined a PRL-signalling cascade in W53 cells, involving Src kinases/PI3K/Akt/FKHRL1-GSK3, that mediates stimulation of c-Myc expression.

Src mediates prolactin-dependent proliferation of T47D and MCF7 cells via the activation of focal adhesion kinase/Erk1/2 and phosphatidylinositol 3-kinase pathways.

Prolactin (PRL) stimulates breast cancer cell proliferation; however, the involvement of PRL-activated signaling molecules in cell proliferation is not fully established. Here we studied the role of c-Src on PRL-stimulated proliferation of T47D and MCF7 breast cancer cells. We initially observed that PRL-dependent activation of focal adhesion kinase (Fak), Erk1/2, and cell proliferation was mediated by c-Src in T47D cells, because expression of a dominant-negative form of c-Src (SrcDM, K295A/Y527F) blocked the PRL-dependent effects. The Src inhibitor PP1 abrogated PRL-dependent in vivo activation of Fak, Erk1/2, p70S6K, and Akt and the proliferation of T47D and MCF7 cells; Janus kinase 2 (Jak2) activation was not affected. However, in vitro, Fak and Jak2 kinases were not directly inhibited by PP1, demonstrating the effect of PP1 on c-Src kinase as an upstream activator of Fak. Expression of Fak mutant Y397F abrogated PRL-dependent activation of Fak, Erk1/2, and thymidine incorporation, but had no effect on p70S6K and Akt kinases. MAPK kinase 1/2 (Mek1/2) inhibitor PD184352 blocked PRL-induced stimulation of Erk1/2 and cell proliferation; however, p70S6K and Akt activation were unaffected. The phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 abolished cell proliferation and activation of p70S6K and Akt; however, PRL-dependent activation of Erk1/2 was not modified. Moreover, we show that both c-Src/PI3K and c-Src/Fak/Erk1/2 pathways are involved in the up-regulation of c-myc and cyclin d1 expression mediated by PRL. The previous findings suggest the existence of two PRL-dependent signaling cascades, initiated by the c-Src-mediated activation of Fak/Erk1/2 and PI3K pathways that, subsequently, control the expression of c-Myc and cyclin D1 and the proliferation of T47D and MCF7 breast cancer cells.

Cyr61 as mediator of Src signaling in triple negative breast cancer cells.

SFKs are involved in tumorigenesis and metastasis. Here we analyzed c-Src contribution to initial steps of metastasis by tetracycline-dependent expression of a specific shRNA-c-Src, which suppressed c-Src mRNA and protein levels in metastatic MDA-MB-231 cells. c-Src suppression did not alter cell proliferation or survival, but it significantly reduced anchorage-independent growth. Concomitantly with diminished tyrosine-phosphorylation/activation of Fak, caveolin-1, paxillin and p130CAS, c-Src depletion also inhibited cellular migration, invasion and transendothelial migration. Quantitative proteomic analyses of the secretome showed that Cyr61 levels, which were detected in the exosomal fraction, were diminished upon shRNA-c-Src expression. In contrast, Cyr61 expression was unaltered inside cells. Cyr61 partially colocalized with cis-Golgi gp74 marker and with exosomal marker CD63, but c-Src depletion did not alter their cellular distribution. In SUM159PT cells, transient c-Src suppression also reduced secreted exosomal Cyr61 levels. Furthermore, conditional expression of a c-Src dominant negative mutant (SrcDN, c-Src-K295M/Y527F) in MDA-MB-231 and in SUM159PT diminished secreted Cyr61 as well. Cyr61 transient suppression in MDA-MB-231 inhibited invasion and transendothelial migration. Finally, in both MDA-MB-231 and SUM159PT, a neutralizing Cyr61 antibody restrained migration. Collectively, these results suggest that c-Src regulates secreted proteins, including the exosomal Cyr61, which are involved in modulating the metastatic potential of triple negative breast cancer cells.

Protein kinase Akt/PKB phosphorylates heme oxygenase-1 in vitro and in vivo.

Heme oxygenase-1 (HO-1) is a stress response protein that protects cells against diverse noxious stimuli. Although regulation of HO-1 occurs mainly at the transcriptional level, its posttranslational modifications remain unexplored. We have identified a putative consensus sequence for phosphorylation by Akt/PKB of HO-1 at Ser188. Recombinant human and rat HO-1, but not mutant HO-1(S188A), are phosphorylated in vitro by Akt/PKB. Isotopic 32P-labeling of HEK293T cells confirmed that HO-1 is a phosphoprotein and that the basal HO-1 phosphorylation is increased by Akt1 activation. HO-1(S188D), a single point mutant equivalent to the phosphorylated protein, exhibited over 1.6-fold higher activity than wild type HO-1. Fluorescence resonance energy transfer (FRET) studies indicated that HO-1(S188D) bound to cytochrome P450 reductase (CPR) and biliverdin reductase (BVR) with a slightly lower Kd than wild-type HO-1. Although the changes in activity are small, this study provides the first evidence for a role of the survival kinase Akt in the regulation of HO-1.

Effect of metamizol on promyelocytic and terminally differentiated granulocytic cells. Comparative analysis with acetylsalicylic acid and diclofenac.

Metamizol is an analgesic and antipyretic agent that can induce agranulocytosis in certain patients. However, its effects on granulocyte viability and differentiation have been poorly evaluated. Here we analysed the effects of metamizol and its active metabolite, 4-methylaminoantipyrine (MAA), on the viability of HL60 promyelocytes and their dimethyl sulphoxide-induced differentiated granulocytes. Metamizol and MAA at 75 microM (above the peak of plasmatic concentration after 2g intake) did not alter granulocytic differentiation of HL60 cells. Only at concentrations above 100 microM, well over the pharmacological range, metamizol-induced apoptosis in about 30% of the HL60 promyelocytes, while HL60-granulocytic terminally differentiated cells were more resistant to this apoptotic action. When the effects of metamizol were compared with those of acetylsalicylic acid (ASA) and diclofenac on cell viability, at equivalent concentrations used in analgesic and antipyretic therapy (75 microM for metamizol, and ASA and 3 microM for diclofenac) their apoptotic effects were similar. Again, the HL60 promyelocytes were more sensitive to apoptosis than granulocytic differentiated cells, as measured by the percentage of sub-G(1) cells detected by flow cytometry and by determination of caspase activity as a function of poly(ADP-ribose) polymerase cleavage. Furthermore, when human blood-derived granulocytes were treated with metamizol, MAA, and ASA at 75 microM or diclofenac at 3 microM, less than 10% of apoptotic granulocytes were detected, whereas at toxicological/suprapharmacological concentrations (10mM), about 90% of granulocytes were apoptotic. These results demonstrate that metamizol, MAA, ASA, and diclofenac, at pharmacological concentrations, neither affect the granulocytic differentiation process nor induce relevant apoptosis on terminally differentiated granulocytes.

Diacylglycerol kinase α promotes 3D cancer cell growth and limits drug sensitivity through functional interaction with Src.

Diacylglycerol kinase (DGK)α converts diacylglycerol to phosphatidic acid. This lipid kinase sustains survival, migration and invasion of tumor cells, with no effect over untransformed cells, suggesting its potential as a cancer-specific target. Nonetheless the mechanisms that underlie DGKα specific contribution to cancer survival have not been elucidated. Using three-dimensional (3D) colon and breast cancer cell cultures, we demonstrate that DGKα upregulation is part of the transcriptional program that results in Src activation in these culture conditions. Pharmacological or genetic DGKα silencing impaired tumor growth in vivo confirming its function in malignant transformation. DGKα-mediated Src regulation contributed to limit the effect of Src inhibitors, and its transcriptional upregulation in response to PI3K/Akt inhibitors resulted in reduced toxicity. Src oncogenic properties and contribution to pharmacological resistance have been linked to its overactivation in cancer. DGKα participation in this central node helps to explain why its pharmacological inhibition or siRNA-mediated targeting specifically alters tumor viability with no effect on untransformed cells. Our results identify DGKα-mediated stabilization of Src activation as an important mechanism in tumor growth, and suggest that targeting this enzyme, alone or in combination with other inhibitors in wide clinical use, could constitute a treatment strategy for aggressive forms of cancer.

Src kinases catalytic activity regulates proliferation, migration and invasiveness of MDA-MB-231 breast cancer cells.

SFKs are frequently deregulated in cancer where they control cellular proliferation, migration, survival and metastasis. Here we study the role of SFKs catalytic activity in triple-negative/basal-like and metastatic human breast cancer MDA-MB-231 cells employing three well-established inhibitors: Dasatinib, PP2 and SU6656. These compounds inhibited migration and invasion. Concomitantly, they reduced Fak, paxillin, p130CAS, caveolin-1 phosphorylation and altered cytoskeletal structures. They also inhibited cell proliferation, but in different manners. Dasatinib and PP2 increased p27(Kip1) expression and reduced c-Myc levels, restraining G1­S transition. In contrast, SU6656 did not modify p27(Kip1) expression, slightly altered c-Myc levels and generated polyploid multinucleated cells, indicating inhibition of cytokinesis. These later effects were also observed in SYF fibroblasts, suggesting a SFKs-independent action. ZM447439, an Aurora B kinase inhibitor, produced similar cell cycle and morphological alterations in MDA-MB-231 cells, indicating that SU6656 blocked Aurora B kinase. This was confirmed by inhibition of histone H3 phosphorylation, the canonical Aurora B kinase substrate. Furthermore, hierarchical clustering analysis of gene expression profiles showed that SU6656 defined a set of genes that differed from Dasatinib and PP2. Additionally, Gene Set Enrichment Analyses revealed that SU6656 significantly reduces the Src pathway. Together, these results show the importance of SFKs catalytic activity for MDA-MB-231 proliferation, migration and invasiveness. They also illustrate that SU6656 acts as dual SFKs and Aurora B kinase inhibitor, suggesting its possible use as a therapeutic agent in breast cancer.

Studying the regulation of MAP Kinase by MAP Kinase phosphatases in vitro and in cell systems.

Signaling through MAPK pathways involves a network of activating kinases and inactivating phosphatases. While single MAPK kinases account for specific activation of the distinct MAPKs, inactivation of MAPKs by phosphatases involves a wider spectrum of enzymes, with phosphatases from distinct families displaying specificity toward MAPKs. The dual-specificity family of MAPK phosphatases, MKPs, constitutes the major group of MAPK inactivating phosphatases. MKPs are widely expressed, in a tissue- and development-regulated manner, and the control of their expression and function is crucial for the regulation of MAPK signaling. Here, we present three methods to analyze the regulation of MAPKs by MKPs, using transient and stable-inducible MKP overexpression cell systems and in vitro phosphatase experiments.

Phosphorylation of serine 11 and serine 92 as new positive regulators of human Snail1 function: potential involvement of casein kinase-2 and the cAMP-activated kinase protein kinase A.

Snail1 is a major factor for epithelial-mesenchymal transition (EMT), an important event in tumor metastasis and in other pathologies. Snail1 is tightly regulated at transcriptional and posttranscriptional levels. Control of Snail1 protein stability and nuclear export by GSK3beta phosphorylation is important for Snail1 functionality. Stabilization mechanisms independent of GSK3beta have also been reported, including interaction with LOXL2 or regulation of the COP9 signalosome by inflammatory signals. To get further insights into the role of Snail1 phosphorylation, we have performed an in-depth analysis of in vivo human Snail1 phosphorylation combined with mutational studies. We identify new phosphorylation sites at serines 11, 82, and 92 and confirmed previously suggested phosphorylations at serine 104 and 107. Serines 11 and 92 participate in the control of Snail1 stability and positively regulate Snail1 repressive function and its interaction with mSin3A corepressor. Furthermore, serines 11 and 92 are required for Snail1-mediated EMT and cell viability, respectively. PKA and CK2 have been characterized as the main kinases responsible for in vitro Snail1 phosphorylation at serine 11 and 92, respectively. These results highlight serines 11 and 92 as new players in Snail1 regulation and suggest the participation of CK2 and PKA in the modulation of Snail1 functionality.

A non-catalytic function of the Src family tyrosine kinases controls prolactin-induced Jak2 signaling.

The cytokine prolactin (PRL) plays important roles in the proliferation and differentiation of the mammary gland and it has been implicated in tumorigenesis. The prolactin receptor (PRLR) is devoid of catalytic activity and its mitogenic response is controlled by cytoplasmic tyrosine kinases of the Src (SFK) and Jak families. How PRLR uses these kinases for signaling is not well understood. Previous studies indicated that PRLR-induced Jak2 activation does not require SFK catalytic activity in favor of separate signaling operating on this cellular response. Here we show that, nevertheless, PRLR requires Src-SH2 and -SH3 domains for Jak2 signaling. In W53 lymphoid cells, conditional expression of two c-Src non-catalytic mutants, either SrcK295M/Y527F or SrcK, whose SH3 and SH2 domains are exposed, controls Jak2/Stat5 activation by recruiting Jak2, avoiding its activation by endogenous active SFK. In contrast, the kinase inactive SrcK295M mutant, with inaccessible SH3 and SH2 domains, does not. Furthermore, all three mutants attenuate PRLR-induced Akt and p70S6K activation. Accordingly, PRLR-induced Jak2/Stat5 signaling is inhibited in MCF7 breast cancer cells by Src depletion, expression of SrcK295M/Y527F or active Src harboring an inactive SH2 (SrcR175L) or SH3 domain (SrcW118A). Finally, Jak2/Stat5 pathway is also reduced in Src-/- mice mammary glands. We thus conclude that, in addition to Akt and p70S6K, SFK regulate PRLR-induced Jak2 signaling through a kinase-independent mechanism.