Biomedical subjects

Hallgeir Rui

Publications and source records attributed to Hallgeir Rui.

18 recordsLinked to original sources

PubMed2006

Caveolin-1, mammary stem cells, and estrogen-dependent breast cancers.

Estrogen exposure is considered a significant risk factor for breast cancer development. Estrogen receptor (ER) alpha is expressed at low levels in normal epithelia, and its expression is dramatically up-regulated as transformation progresses during mammary hyperplasia and adenocarcinoma development. The mechanism(s) driving ERalpha up-regulation during mammary tumorigenesis remains unclear. Caveolin-1 (Cav-1) is the structural protein of plasmalemmal invaginations, termed caveolae, which functions as a tumor suppressor gene. Interestingly, Cav-1 dominant-negative mutations are exclusively found in ERalpha-positive breast cancer samples. In support of these clinical findings, ERalpha expression is increased in Cav-1 (-/-) null mammary epithelia, and estrogen stimulation further enhances the growth of Cav-1-deficient three-dimensional epithelial structures. These phenotypes correlate with augmented levels of cyclin D1. In addition, Cav-1 gene inactivation induces the accumulation of a cell population with the characteristics of adult mammary stem cells. Primary cultures of Cav-1 (-/-) mammary epithelial cells exhibit premalignant changes, such as abnormal lumen formation, epidermal growth factor-independent growth, defects in cell substrate attachment, and increased cell invasiveness. Thus, Cav-1 gene inactivation promotes premalignant alterations in mammary epithelia and induces increased ERalpha expression levels and the up-regulation of cyclin D1. As tumor formation is a multihit process, Cav-1 mutations that occur during the early stages of mammary transformation may be a critical upstream/initiating event leading to increased ERalpha levels.

Adult Stem Cells↗

PubMed2006

A preferential role for STAT5, not constitutively active STAT3, in promoting survival of a human lymphoid tumor.

STATs are believed to play key roles in normal and abnormal cell function. In the present work, we investigated the role of STATs in an IL-2-responsive human lymphoblastic lymphoma-derived cell line, YT. Only STAT3 was found constitutively tyrosine phosphorylated, but not other STATs. Hyperactive STAT3 was not attributable to a pre-existing intermediate affinity IL-2R complex and/or hyperactive Jak activity. Depletion of STAT3 protein expression reduced tumor cell viability with protracted kinetics (72-96 h), while TUNEL assays demonstrated cell death occurred via apoptosis. Interestingly, depletion of STAT5 in this same tumor induced more pronounced cell death compared with STAT3 depletion (24 h). Although IL-2 was able to rescue STAT3-depleted cells from death, it could not compensate for the loss of STAT5. To determine the prosurvival function of STAT3 vs STAT5 within the same tumor model, genes were profiled in STAT3- or STAT5-depleted YT cells by apoptosis-specific microarrays. Several differentially expressed genes were identified. Interestingly, those genes involved in NF-kappaB regulation, such as TNFR-associated factors 2 and 5 and B cell leukemia/lymphoma 10, were readily decreased upon STAT5, but not STAT3, depletion as validated by quantitative RT-PCR. These results suggest that STAT5 and, to a lesser extent, hyperactive STAT3 provide preferential and critical cell survival signals for certain human lymphoid tumors, indicating that nonhyperactive STATs should be considered as therapeutic targets for abrogating tumorigenesis.

Apoptosis↗

PubMed2006

Stromal and epithelial caveolin-1 both confer a protective effect against mammary hyperplasia and tumorigenesis: Caveolin-1 antagonizes cyclin D1 function in mammary epithelial cells.

Here, we investigate the role of caveolin-1 (Cav-1) in breast cancer onset and progression, with a focus on epithelial-stromal interactions, ie, the tumor microenvironment. Cav-1 is highly expressed in adipocytes and is abundant in mammary fat pads (stroma), but it remains unknown whether loss of Cav-1 within mammary stromal cells affects the differentiated state of mammary epithelia via paracrine signaling. To address this issue, we characterized the development of the mammary ductal system in Cav-1-/- mice and performed a series of mammary transplant studies, using both wild-type and Cav-1-/- mammary fat pads. Cav-1-/- mammary epithelia were hyperproliferative in vivo, with dramatic increases in terminal end bud area and mammary ductal thickness as well as increases in bromodeoxyuridine incorporation, extracellular signal-regulated kinase-1/2 hyperactivation, and up-regulation of STAT5a and cyclin D1. Consistent with these findings, loss of Cav-1 dramatically exacerbated mammary lobulo-alveolar hyperplasia in cyclin D1 Tg mice, whereas overexpression of Cav-1 caused reversion of this phenotype. Most importantly, Cav-1-/- mammary stromal cells (fat pads) promoted the growth of both normal mammary ductal epithelia and mammary tumor cells. Thus, Cav-1 expression in both epithelial and stromal cells provides a protective effect against mammary hyperplasia as well as mammary tumorigenesis.

Adipose Tissue↗

PubMed2005

The gammac-cytokine regulated transcription factor, STAT5, increases HIV-1 production in primary CD4 T cells.

Although HIV-1 (HIV) replicates poorly in non-dividing CD4 lymphocytes, resting T cells contribute to the latent reservoir. The gammac-related cytokines reverse this block to HIV infection; however, the molecular mechanisms controlling this process are not understood. We asked whether the gammac-cytokine regulated transcription factor, signal transducer and activator of transcription 5 (STAT5), activates HIV transcription. We identified three regions in the long terminal repeat (LTR) as close matches to the STAT5 consensus-binding site and show that STAT5 binds the LTR during HIV infection. Expression of Janus kinase 3 (JAK3) or STAT5 in primary human CD4 T cells activated LTR transcription, while transactivation-incompetent dominant-negative STAT5 inhibited JAK3-induced LTR activity and infection of activated HIV-producing CD4 T-cells. In addition, overexpression of STAT5 increased virus production in unstimulated primary T cells - both the number of p24+ cells and their level of p24 production - suggesting that STAT5 promotes a permissive state for HIV infection. These data may have implications for regulation of latency and therapeutic strategies for control of HIV disease.

Binding Sites↗

PubMed2005

Evaluation of genome-wide chromatin library of Stat5 binding sites in human breast cancer.

BACKGROUND: There is considerable interest in identifying target genes and chromatin binding sites for transcription factors in a genome-wide manner. Such information may become useful in diagnosis and treatment of disease, drug target identification, and for prognostication. In cancer diagnosis, patterns of transcription factor binding to specific regulatory chromatin elements are expected to complement and enhance current diagnostic predictions of tumor behavior based on protein and mRNA analyses. Signal transducer and activator of transcription-5 (Stat5) is a cytokine-activated transcription factor implicated in growth and progression of many malignancies, including hematopoietic, prostate, and breast cancer. We have explored immunoaffinity purification of Stat5-bound chromatin from breast cancer cells to identify Stat5 target sites in an unbiased, genome-wide manner. RESULTS: In this report, we evaluate the efficacy of a Stat5-bound chromatin library to identify valid Stat5 chromatin binding sites within the oncogenome of T-47D human breast cancer cells. A general problem with cloning of immunocaptured, transcription factor-bound chromatin fragments is contamination with non-specific chromatin. However, using an optimized strategy, five out of ten randomly selected clones could be experimentally verified to bind Stat5 both in vitro and in vivo as tested by electrophoretic mobility shift assay and chromatin immunoprecipitation, respectively. While there was no binding to fragments lacking a Stat5 consensus binding sequence, presence of a Stat5 binding sequence did not assure binding. CONCLUSION: A chromatin library coupled with experimental validation may productively identify novel in vivo Stat5 chromatin binding sites in cancer, including abnormal regulatory sites in tumor-specific neochromatin.

Binding Sites↗

PubMed2005

Stat5 promotes homotypic adhesion and inhibits invasive characteristics of human breast cancer cells.

Signal transducer and activator of transcription-5 (Stat5) mediates prolactin (PRL)-induced differentiation and growth of breast epithelial cells. We have recently identified active Stat5 as a tumor marker of favorable prognosis in human breast cancer, and determined that Stat5 activation is lost during metastatic progression. Here we provide novel evidence for an invasion-suppressive role of Stat5 in human breast cancer. Activation of Stat5 by PRL in human breast cancer lines was associated with increased surface levels of the invasion-suppressive adhesion molecule E-cadherin in vitro and in xenotransplant tumors in vivo. Inducible E-cadherin was blocked by dominant-negative (Dn) Stat5 or Dn-Jak2, but not by Dn-Stat3. Further experimental data indicated a role of Stat5 as a coordinate regulator of additional invasion-related characteristics of human breast cancer cells, including cell surface association of beta-catenin, homotypic cell clustering, invasion through Matrigel, cell migration, and matrix metalloproteinase activity. A role of Stat5 as a suppressor of breast cancer invasion and metastatic progression provides a biological mechanism to explain the favorable prognosis associated with active Stat5 in human breast cancer.

Animals↗

PubMed2005

Ultrahigh density microarrays of solid samples.

We present a sectioning and bonding technology to make ultrahigh density microarrays of solid samples, cutting edge matrix assembly (CEMA). Maximized array density is achieved by a scaffold-free, self-supporting construction with rectangular array features that are incrementally scalable. This platform technology facilitates arrays of >10,000 tissue features on a standard glass slide, inclusion of unique sample identifiers for improved manual or automated tracking, and oriented arraying of stratified or polarized samples.

Equipment Design↗

PubMed2005

Creating tissue microarrays by cutting-edge matrix assembly.

Tissue microarrays have become widely adopted for effective parallel in situ analysis of hundreds of tissues placed onto single slides. Traditionally, tissue core punches are transferred into predrilled holes within a scaffold block of paraffin or other material, and sectioned transversely by a microtome to generate array sections. While core-based arraying has greatly advanced tissue analyses, some of the limitations include restricted feature sizes and numbers, variable core depths of unpredictable tissue quality and inability to array thin-walled, stratified tissue samples such as intestines, vessels or skin. Overcoming these limitations, the authors have developed a practical arraying method that combines serial cutting and edge-to-edge bonding of samples to assemble a scaffold-free array matrix -- cutting-edge matrix assembly. Using cutting-edge matrix assembly, the authors have successfully placed more than 10,000 individual tissue pieces on a single histology glass slide. The potential biomedical utility and ongoing efforts to further develop the assembly technology and analysis of high-density cutting-edge matrix assembly tissue microarrays is discussed.

Equipment Design↗

PubMed2004

Signal transducer and activator of transcription-5 activation and breast cancer prognosis.

PURPOSE: Transcription factor signal transducer and activator of transcription-5 (Stat5) promotes breast epithelial cell differentiation. We retrospectively analyzed whether levels of active Stat5 in breast cancer were linked to clinical outcome. MATERIALS AND METHODS: Immunohistochemistry was used to detect active, tyrosine-phosphorylated Stat5 in paraffin-embedded breast cancer specimens from three archival tissue microarray materials A, B, and C. Material A included 19 healthy human breast tissues and a progression series of primary lymph node-negative, primary lymph node-positive, and metastatic breast cancer (n = 400). Materials B (n = 785) and C (n = 570) represented two independent arrays of unselected primary breast cancer specimens with clinical follow-up data. RESULTS: Material A demonstrated that Stat5 activation, but not Stat5 protein expression, was gradually lost during cancer progression, with detectable activation in 100% of healthy breast specimens compared with less than 20% of node-positive breast cancers and metastases. Stat5 activation in tumors of material B was associated with favorable prognosis. This observation was confirmed and extended in material C to include both breast cancer-specific survival and disease-free survival. Stat5 activation remained an independent prognostic marker after adjusting for patient age, tumor size, histological grade, estrogen receptor, progesterone receptor, and Her2/neu status by Cox multivariate analysis (hazard ratio, 2.0; P =.029). Stat5 activation was a particularly favorable marker in the lymph node-negative breast cancer subpopulation (hazard ratio, 7.5; P =.003). CONCLUSION: In our study, active Stat5 distinguishes breast cancer patients with favorable prognosis, and may be a useful marker for selection of more individualized treatment, especially in localized disease. These findings require confirmation in a large prospective study.

Adult↗

PubMed2004

Generation of a conditional knockout allele for the Janus kinase 2 (Jak2) gene in mice.

To study biologically relevant functions of the Janus kinase 2 (Jak2) in multiple cytokine and hormone receptor signal transduction pathways, we generated a conditional knockout (floxed) allele of this gene by placing loxP sites around the first coding exon of Jak2. Homozygous floxed animals developed normally and exhibited no phenotypic abnormalities. The conversion of the floxed allele into a null mutation was achieved by transmitting the targeted allele through the female germline of MMTV-Cre (line A) mice. Embryos that carry two Jak2 null alleles died around midgestation and exhibited impaired definitive erythropoiesis, which is a hallmark of Jak2 deficiency reported previously in conventional knockouts. This observation suggested that the Cre-mediated deletion of the first coding exon results in a true null mutation that is incapable of mediating signals through the erythropoietin receptor. Using mouse embryonic fibroblasts derived from Jak2 null embryos and their wildtype littermate controls, we demonstrated that Jak2-deficiency decouples growth hormone-receptor signaling from its downstream mediators, the signal transducer and activator of transcription (Stat) 5a and 5b.

Animals↗

PubMed2004

Impaired alveologenesis and maintenance of secretory mammary epithelial cells in Jak2 conditional knockout mice.

Jak2 is a hormone-receptor-coupled kinase that mediates the tyrosine phosphorylation and activation of signal transducers and activators of transcription (Stat). The biological relevance of Jak2-Stat signaling in hormone-responsive adult tissues is difficult to investigate since Jak2 deficiency leads to embryonic lethality. We generated Jak2 conditional knockout mice to study essential functions of Jak2 during mammary gland development. The mouse mammary tumor virus-Cre-mediated excision of the first coding exon resulted in a Jak2 null mutation that uncouples signaling from the prolactin receptor (PRL-R) to its downstream mediator Stat5 in the presence of normal and supraphysiological levels of PRL. Jak2-deficient females were unable to lactate as a result of impaired alveologenesis. Unlike Stat5a knockouts, multiple gestation cycles could not reverse the Jak2-deficient phenotype, suggesting that neither other components of the PRL-R signaling cascade nor other growth factors and their signal transducers were able to compensate for the loss of Jak2 function to activate Stat5 in vivo. A comparative analysis of Jak2-deficient mammary glands with transplants from Stat5a/b knockouts revealed that Jak2 deficiency also impairs the pregnancy-induced branching morphogenesis. Jak2 conditional mutants therefore resemble PRL-R knockouts more closely, which suggested that Jak2 deficiency might affect additional PRL-R downstream mediators other than Stat5a and Stat5b. To address whether Jak2 is required for the maintenance of PRL-responsive, differentiating alveolar cells, we utilized a transgenic strain that expresses Cre recombinase under regulatory elements of the whey acidic protein gene (Wap). The Wap-Cre-mediated excision of Jak2 resulted in a negative selection of differentiated alveolar cells, suggesting that Jak2 is required not only for the proliferation and differentiation of alveolar cells but also for their maintenance during lactation.

Animals↗

PubMed2003

Inhibition of transcription factor Stat5 induces cell death of human prostate cancer cells.

Identifying regulators of prostate cancer cell survival may lead to new therapeutic strategies for prostate cancer. We now report prevalent activation of transcription factor Stat5 in human prostate cancer and provide novel evidence that blocking activation of Stat5 in human prostate cancer cells leads to extensive cell death. Specifically, Stat5 was activated in 65% of human prostate cancer specimens examined based on nuclear location of tyrosine phosphorylated Stat5. Adenoviral gene delivery of a dominant-negative Stat5 mutant (DNStat5), but not wild-type Stat5, induced cell death of both the androgen-independent human prostate cancer cell line CWR22Rv and the androgen-sensitive LnCap cell line. Endogenous Stat5 was active in both CWR22Rv and LnCap cells. In contrast, only low levels of inactive Stat5 proteins were detected in the PC-3 cell line, which correlated with resistance to DNStat5-induced cell death. In CWR22Rv and LnCap cells, inhibition of Stat5 by expression of DNStat5 induced apoptotic cell death as judged from morphological changes, DNA fragmentation, and caspase-3 activation with evidence of a caspase-9-dependent mechanism. We propose that blocking Stat5 function may represent a novel therapeutic approach for prostate cancer.

Adenoviridae↗

PubMed2003

STAT5a activation mediates the epithelial to mesenchymal transition induced by oncogenic RhoA.

The involvement of Rho GTPases in signal transduction pathways leading to transcription activation is one of the major roles of this family of GTPases. Thus, the identification of transcription factors regulated by Rho GTPases and the understanding of the mechanisms of their activation and its biological outcome are of great interest. Here, we provide evidence that Rho GTPases modulate Stat5a, a transcription factor of the family of signal transducers and activators of transcription. RhoA triggers tyrosine phosphorylation (Y696) of Stat5a via a JAK2-dependent mechanism and promotes DNA-binding activity of Stat5a. Tyrosine phosphorylation of Stat5a is also stimulated physiologically by lysophosphatidic acid (LPA) in a Rho-dependent manner. Simultaneously, RhoA reduces serine phosphorylation of Stat5a at both serine residues S726 and S780, resulting in a further increase of activity as defined by mutagenesis experiments. Furthermore, serine dephosphorylation of Stat5a by RhoA does not take place by down-modulation of either JNK1, MEK1, or p38 MAP kinases, as determined by transfection experiments or chemical inhibition of both MEK1, p38, and JNK serine kinases. Thus, RhoA regulates Stat5a via tyrosine phosphorylation and via a yet to be determined novel down-modulating pathway that involves serine dephosphorylation. Finally, we provide evidence for a role of Stat5a in RhoA-induced epithelial-to-mesenchymal transition with concomitant increase in vimentin expression, E-cadherin down-regulation, and cell motility.

DNA-Binding Proteins↗

PubMed2002

Prostaglandin-E2 enhances EPO-mediated STAT5 transcriptional activity by serine phosphorylation of CREB.

Erythroid colony formation in response to erythropoietin (EPO) stimulation is enhanced by costimulating the cells with prostaglandin-E2 (PGE2). The present study further analyzed the underlying mechanisms and demonstrated that EPO-mediated STAT5 transactivation in the erythroid AS-E2 cell line was enhanced 6-fold by PGE2 (10 microM), without affecting the STAT5 tyrosine phosphorylation or STAT5-DNA binding. Moreover, the PGE2-enhancing effect was independent of STAT5 serine phosphorylation. In AS-E2 cells STAT5 is constitutively phosphorylated on Ser780 (STAT5A) and EPO-dependently phosphorylated on Ser726/731 (STAT5A/STAT5B), but overexpression of STAT5 serine mutants did not affect STAT5 transactivation. In addition, PGE2 did not affect STAT5 serine phosphorylation. Instead, the stimulatory effect of PGE2 on STAT5 signaling could be mimicked by dibutyryl-cyclic adenosine monophosphate (cAMP) and the phosphodiesterase inhibitor IBMX, suggesting that the effect was mediated by cAMP. Activation of the cAMP pathway resulted in cAMP-response element binding protein (CREB) phosphorylation, which was sustained in the presence of EPO plus PGE2 and transient on EPO stimulation alone. The costimulatory effect of PGE2 on EPO-mediated STAT5 transactivation was inhibited by overexpression of serine-dead CREB or protein kinase A (PKA) inhibitor (PKI), in contrast to EPO-mediated transactivation, which was PKA independent. Furthermore, CREB-binding protein (CBP)/p300 was shown to be involved in EPO-mediated STAT5 transactivation, and a CBP mutant with increased affinity for CREB resulted in an additional enhancement of the PGE2 effect. Finally, we demonstrated that the STAT5 target genes Bcl-X, SOCS2, and SOCS3 were up-regulated by costimulation with PGE2. In summary, these studies demonstrate that PGE2 enhancement of EPO-induced STAT5 transactivation is mediated by the cAMP/PKA/CREB pathway.

Cell Line↗

PubMed2002

Role of tyrosine kinase Jak2 in prolactin-induced differentiation and growth of mammary epithelial cells.

Genetic studies in mice have established a critical role for prolactin receptors and transcription factor Stat5 in mammary gland differentiation. However, the enzymatic coupling between prolactin receptors and Stat5 in this process has not been established. In addition to Jak2, several other tyrosine kinases reportedly also are associated with prolactin receptors and may phosphorylate Stat5. Because Jak2 null mice die in utero, we targeted Jak2 in an ex vivo model of prolactin-induced mammary epithelial cell differentiation to determine the role of Jak2 in regulation of cell differentiation and growth. Two independent targeting strategies were used to suppress Jak2 in immortalized HC11 mouse mammary epithelial cells: 1) stable expression of a specific Jak2 antisense construct and 2) adenoviral delivery of a dominant-negative Jak2 gene. We now demonstrate that Jak2 is essential for prolactin-induced differentiation and activation of Stat5 in normal mouse mammary epithelial cells. Furthermore, suppression of Jak2 in HC11 cells was associated with constitutive activation of oncoprotein Stat3 and a hyperproliferative phenotype characterized by increased mitotic rate, reduced apoptosis, and reduced contact inhibition. Collectively, our data suggest that Jak2 is differentiation-inducing and growth-inhibitory in normal mammary epithelial cells, observations that may shed new light on the role of the Jak2-Stat5 pathway in breast cancer.

Adenoviridae↗

PubMed2002

PRL signal transduction in the epithelial compartment of rat prostate maintained as long-term organ cultures in vitro.

Using long-term organ cultures of rat prostate tissue explants, we previously demonstrated that PRL both stimulates proliferation and acts as an androgen-independent suppressor of apoptosis in prostate epithelial cells, leading to epithelial hyperplasia. In this work we delineate intracellular signaling molecules activated by PRL in prostate tissue to identify candidate signaling proteins that are responsible for maintaining survival and proliferation of prostate epithelium in androgen-deprived growth environment. We now show that signal transducer and activator of transcription-5a (Stat5a) and Stat5b become tyrosine phosphorylated in response to PRL stimulation in rat prostate using prostate organ culture as an experimental model. Stat5 was translocated to the nuclei of epithelial cells of prostate tissue as demonstrated by immunohistochemistry. Furthermore, EMSA showed PRL-inducible binding of Stat5a homodimers and Stat5a/5b heterodimers to the PRL response element of the beta-casein gene promoter. Signaling molecules Stat3, Stat1, MAPK, or protein kinase B, which can be activated by PRL in other target cells, were not activated by PRL in prostate tissue. Furthermore, we show that Stat5a and Stat5b are continuously phosphorylated in rat prostate in vivo, although they are expressed to varying degree in separate lobes of rat prostate. Collectively, our results suggest that PRL signaling in rat prostate tissue is primarily transduced via Stat5a and Stat5b. The Stat5 pathway represents one candidate signaling mechanism, used by PRL and possibly other growth factors and cytokines, that supports the viability of prostate epithelial cells during long-term androgen deprivation.

Animals↗

PubMed2002

Basal activation of transcription factor signal transducer and activator of transcription (Stat5) in nonpregnant mouse and human breast epithelium.

Transcription factor Stat5 (signal transducer and activator of transcription) is essential for PRL-induced terminal differentiation of mouse mammary epithelial cells during pregnancy and lactation and has been implicated in mammary tumorigenesis. A new and sensitive immunological method to detect active, tyrosine phosphorylated Stat5 in situ revealed that Stat5 is continuously activated in luminal epithelial cells of mouse and human breast, not only during pregnancy and lactation, but also outside of pregnancy. Examination of virgin Stat5a or Stat5b null mice suggested that Stat5a was the primary isoform activated in mammary epithelial cells. Basal activation of Stat5 in mammary epithelium of virgin wild-type mice was continuous throughout estrous cycle and was also detected in 17 of 17 normal human breast tissue specimens analyzed. PRL was identified as the principal factor maintaining basal activation of Stat5 in mammary epithelium of nonpregnant mice based on several lines of evidence. First, administration of PRL, but not GH or epidermal growth factor, uniformly enhanced basal activation of Stat5 in luminal mammary epithelial cells. Second, hypophysectomy disrupted basal activation of Stat5, an effect that was completely reversed by administration of PRL, but only partially by GH. Third, knock-out of the PRL receptor gene markedly reduced basal activation of Stat5, an effect that was maintained in a normalized endocrine environment after transplanting PRL receptor null mammary epithelium into wild-type mice. Continuous activation of Stat5 indicates a role of this transcription factor in normal, nonpregnant breast epithelial cells, and may shed new light on Stat5 involvement in breast tumor promotion.

Animals↗

PubMed2002

Interleukin-2 family cytokines stimulate phosphorylation of the Pro-Ser-Pro motif of Stat5 transcription factors in human T cells: resistance to suppression of multiple serine kinase pathways.

Signal transducer and activator of transcription (Stat)5a and Stat5b are critical for normal immune function. Progression of T cells through G(1)-S phase of cell cycle requires T cell receptor (TCR)- and/or cytokine-inducible tyrosine phosphorylation of Stat5a/b. Stat5a/b may also, in a cell-dependent manner, be constitutively or cytokine-inducibly phosphorylated on a Pro-Ser-Pro (PSP) motif located within the transcriptional activation domain. Phosphorylation of the PSP motif is needed for maximal transcriptional activation by Stat5, at least in certain promoter contexts. The basal and cytokine-inducible serine phosphorylation state of Stat5a/b has not been determined in T cells. Using primary human T cells and T lymphocytic cell lines coupled with novel phospho-specific antibodies to this conserved phosphoserine motif in Stat5a or Stat5b, we report that: Stat5a and Stat5b were unphosphorylated on the PSP motif under basal conditions and became markedly phosphorylated in response to several T cell growth factor stimuli, including interleukin (IL)-2, -7, -9, and -15 and phorbol ester 12-myristate 13-acetate but not TCR engagement; inducible Stat5a/b serine phosphorylation differed quantitatively and temporally; and Stat5a/b serine phosphorylation was, in contrast to inducible Stat3 serine phosphorylation, insensitive to inhibitors of mitogen-activated protein kinase, phosphatidylinositol-3 kinase, and mammalian target of rapamycin or deletion of Raf-A, -B, or -C by antisense oligonucleotides. We conclude that IL-2 family cytokines tightly control Stat5 serine phosphorylation through a kinase distinct from the Stat3 serine kinase.

Amino Acid Motifs↗