Activation of phosphatidylinositide 3-OH kinase (PI 3-kinase) is implicated in mediating a variety of growth factor-induced responses, among which are the inactivation of glycogen synthase kinase-3 (GSK-3) and the activation of the serine/threonine protein kinase B (PKB). GSK-3 inactivation occurs through phosphorylation of Ser-9, and several kinases, such as protein kinase C, mitogen-activated protein kinase-activated protein kinase-1 (p90 Rsk ), p70 S6kinase , and also PKB have been shown to phosphorylate this site in vitro. In the light of the many candidates to mediate insulin-induced GSK-3 inactivation we have investigated the role of PKB by constructing a PKB mutant that exhibits dominant-negative function (inhibition of growth factor-induced activation of PKB at expression levels similar to wild-type PKB), as currently no such mutant has been reported. We observed that the PKB mutant (PKB-CAAX) acts as an efficient inhibitor of PKB activation and also of insulin-induced GSK-3 regulation. Furthermore, it is shown that PKB and GSK-3 co-immunoprecipitate, indicating a direct interaction between GSK-3 and PKB. An additional functional consequence of this interaction is implicated by the observation that the oncogenic form of PKB, gagPKB induces a cellular relocalization of GSK-3 from the cytosolic to the membrane fraction. Our results demonstrate that PKB activation is both necessary and sufficient for insulin-induced GSK-3 inactivation and establish a linear pathway from insulin receptor to GSK-3. Regulation of GSK-3 by PKB is likely through direct interaction, as both proteins co-immunoprecipitate. This interaction also resulted in a translocation of GSK-3 to the membrane in cells expressing transforming gagPKB.Activation of membrane-bound receptors of both the serpentine and tyrosine kinase classes often results in the activation of the lipid kinase PI 1 3-kinase, of which several different isoforms have been described (1). Activation of PI 3-kinase results in the formation of 3Ј-phosphorylated phosphatidylinositol (PI-3P) lipids (i.e. PI 3,4-P 2 and PI 3,4,5-P 3 ). These lipids were long suspected to perform a second messenger function. However, until recently the nature of the PI-3P binding proteins and their role in cellular signaling remained unsolved.

In fibroblasts, stimulation of receptor tyrosine kinases results in the activation of the extracellular signal‐regulated kinase 2 (ERK2). The major signalling pathway employed by these receptors involves the activation of p21ras and raf‐1 kinase. Here we show that in NIH3T3 and rat‐1 fibroblasts, elevation of the intracellular cAMP level results in the inhibition of ERK2 activation induced by PDGF, EGF and insulin treatment. Analysis of various signalling intermediates shows that cAMP interferes at a site downstream of p21ras, but upstream of raf‐1 kinase. Inhibition by cAMP depends on both the cAMP concentration and the absolute amount of p21ras molecules bound to GTP, suggesting a mechanism of competitive inhibition. Also TPA‐induced, p21ras‐independent, activation of raf‐1 kinase and ERK2 is inhibited by cAMP. We have used the inhibitory effect of cAMP to investigate whether phosphorylation of mSos, a p21ras nucleotide exchange factor, is dependent on the activity of the raf‐1 kinase/ERK2 pathway. We found that phosphorylation of mSos, as monitored by a mobility shift, is delayed with respect to p21ras and ERK2 activation and is inhibited by cAMP in a similar cell type‐ and concentration‐dependent manner as the inactivation of ERK2. These results provide evidence for a model of p21ras‐directed signalling towards ERK2 that feeds back on mSos by regulating its phosphorylation status and that can be negatively modulated by protein kinase A and positively modulated by protein kinase C action.

Tuberculosis (TB) is an escalating global health problem and improved vaccines against TB are urgently needed. HLA-E restricted responses may be of interest for vaccine development since HLA-E displays very limited polymorphism (only 2 coding variants exist), and is not down-regulated by HIV-infection. The peptides from Mycobacterium tuberculosis (Mtb) potentially presented by HLA-E molecules, however, are unknown. Here we describe human T-cell responses to Mtb-derived peptides containing predicted HLA-E binding motifs and binding-affinity for HLA-E. We observed CD8+ T-cell proliferation to the majority of the 69 peptides tested in Mtb responsive adults as well as in BCG-vaccinated infants. CD8+ T-cells were cytotoxic against target-cells transfected with HLA-E only in the presence of specific peptide. These T cells were also able to lyse M. bovis BCG infected, but not control monocytes, suggesting recognition of antigens during mycobacterial infection. In addition, peptide induced CD8+ T-cells also displayed regulatory activity, since they inhibited T-cell proliferation. This regulatory activity was cell contact-dependent, and at least partly dependent on membrane-bound TGF-β. Our results significantly increase our understanding of the human immune response to Mtb by identification of CD8+ T-cell responses to novel HLA-E binding peptides of Mtb, which have cytotoxic as well as immunoregulatory activity.

Expression of p21'n-'7, a dominant negative mutant of p2l' that blocks p2l' activation by growth factors, inhibits activation of extracellular signal-regulated kinase 2 (ERK2) by insulin and platelet-derived growth factor in rat-1 cells [A. M. M. de Vries-Smits, B. M. T. Burgering, S. J. Leevers, C. J. Marshall, and J. L. Bos, Nature (London) 357:602-604, 1992]. Here we report that expression of p21'asAsn-17 does not abolish epidermal growth factor (EGF)-induced phosphorylation of ERK2 in fibroblasts. Since EGF activates p2l' in these cells, this indicates that EGF induces a p21'-independent pathway for the phosphorylation of ERK2 as well. We investigated whether activation of protein kinase C (PKC) or increase in intracellular calcium could be involved in p21a-independent signaling. In rat-i cells, inhibition of either PKC, by prolonged 12-0-tetradecanoylphorbol-13-acetate (TPA) pretreatment, or calcium influx, by ethylene glycol-bis(1-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) pretreatment, did not abolish EGF-induced ERK2 phosphorylation. However, a combined inhibition of both p2l' and calcium influx, but not PKC, resulted in a complete inhibition of EGF-induced ERK2 phosphorylation. In contrast, in Swiss 3T3 cells, inhibition of both p2l' activation and TPA-sensitive PKC, but not calcium influx, inhibited EGF-induced ERK2 phosphorylation. These results demonstrate that in fibroblasts, EGF induces alternative pathways of ERK2 phosphorylation in a cell-type-specific manner.Activation of transmembrane signaling receptors by first messengers generates the formation of second messengers. These second messengers are implicated in the induction of a myriad of intracellular signals of various biochemical constitutions. A common factor of signaling by many mitogenic stimuli is the activation of microtubule-associated protein 2 (MAP2) kinase (for recent reviews, see references 10, 40, and 42). Two forms of MAP2 kinase have been purified from fibroblasts with apparent molecular weights of 42 and 44 kDa. The p42m''K and p44mAPK have been cloned and are also named, respectively, extracellular signal-regulated kinase 2 (ERK2) and 1 (ERK1), to reflect the diversity of signals that can regulate them (3). Activation of both ERK1 and ERK2 requires phosphorylation at neighboring threonine and tyrosine residues and dephosphorylation of activated ERK2 by serine/threonine phosphatases or tyrosine phosphatases results in inactive ERK2 (1, 16). For ERK2, the sites of phosphorylation are located within one tryptic peptide and have been assigned to .Since full activation of ERK2 requires both threonine and tyrosine phosphorylation, it has been suggested that integration of two signaling pathways is required for the activation of the kinase (1). However, it is now clear that ERK2 is part of a kinase/phosphatase cascade in which at least several serine/threonine kinases act upstream of ERK2. The MAP kinase kinase (MAPKK or MEK) that activates ERK2 and phosphorylates the threonine and tyrosine site of ERK2 has recently been clo...

Oosterhuis JW, Kersemaekers AMF, Jacobsen GK, Timmer A, Steyerberg EW, Molier M, van Weeren PC, Stoop H, Looijenga LHJ. Morphology of testicular parenchyma adjacent to germ cell tumours. An interim report. APMIS 2003;111:32-42.A comparative morphological analysis of parenchyma adjacent to testicular germ cell tumours (TGCT) was performed in a series of 181 orchidectomy specimens: 86 with seminomas (Se), 72 with nonseminomatous germ cell tumours (NS) and 23 with combined tumours (CT, which have a Se and a NS component). The following morphological features were semiquantitatively scored: spermatogenesis (modified Johnsen score); amount of tubular atrophy; amount of carcinoma in situ (CIS); amount of intertubular tissue. Absence and presence was scored for the following features: lymphocytic infiltrate surrounding and invading CIS; intratubular seminoma (ISe); intratubular nonseminoma (INS); microlithiasis; diffuse and nodular hyperplasia of Leydig cells; angioinvasiveness; testicular angiopathy. Using non-parametric statistics these features were correlated with each other and with tumour type, tumour size and age of the patient. Se-patients presented at significantly higher age than NS-patients (36 vs 29 years, pΩ0.001). The age of patients with CT (32 years) was in between that of Se-and NS-patients. No correlation was found between patient age and tumour size. Parenchyma adjacent to Se, compared to parenchyma adjacent to NS had the following significant differences: a lower Johnsen score (5.6 vs 7.2, pΩ0.005); less frequent (85% vs 97% of specimens, pΩ0.016) and a lesser amount of CIS (26% vs 32% of tubules, pΩ0.015); more frequent peri-(80% vs 60% of specimens, pΩ0.001) and intratubular (68% vs 30% of specimens, pΩ0.001) lymphocytic infiltrates; more extensive tubular atrophy (36% vs 15% of tubules, pΩ0.001); and a larger area of intertubular tissue (42% vs 34% of parenchyma area, pΩ0.016). The pooled Se and CT had a significantly higher frequency of ISe than the NS (31% vs 17% of specimens, pΩ0.036). With one exception INS was only found adjacent to NS or CT, with a frequency of 16%, and 20% of the specimens, respectively. It was significantly associated with angio-invasiveness. In specimens lacking angio-invasion the frequency of INS was 6%. The correlation of INS with tumour size and patient age was studied in a series of 145 NS and CT (95 from the original series supplemented by 50 newer cases). In this series INS was significantly associated with smaller tumours and younger patients. Extensive tubular atrophy was significantly correlated with higher age, the diagnosis of Se, a low Johnsen score, and the presence of angiopathy. The more tubular atrophy, the less CIS (both in incidence and amount). Inversely, a higher Johnsen score is associated with smaller tumours, the diagnosis of NS or CT, a higher incidence and a larger amount of CIS, and little tubular atrophy. Tubules with mature spermatogenesis were found in 42% of the specimens regardless of tumour type. We conclude that ISe and INS are probably fr...

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Intercellular contacts, mediated by E-cadherin, are essential for germ cell migration and maturation. Furthermore, it has been suggested that decrease or loss of E-cadherin correlates with tumour progression and invasive behaviour. beta-catenin is involved in a number of different processes, including cell--cell interaction when bound to cadherins, and determination of cell fate in pluripotent cells when activated via the Wnt signal-transduction pathway. To shed more light on the role of these factors in normal fetal germ cell development and the pathogenesis of germ cell tumours (GCTs), the present study investigated the presence and localization of E-cadherin and beta-catenin by immunohistochemistry. E-cadherin was only weakly expressed in or absent from fetal germ cells of the second and third trimesters, and was not expressed in carcinoma in situ/intratubular germ cell neoplasia unclassified (CIS/ITGCNU) and gonadoblastoma, the precursor of an invasive GCT in dysgenetic gonads. In GCTs, it was generally not expressed in seminoma and dysgerminoma, but was found in the vast majority of non-seminoma cells. beta-catenin was found in the cytoplasm of fetal germ cells at all gestational ages and in spermatogenesis in post-pubertal testes. It was also present in CIS/ITGCNU and gonadoblastoma. Whereas seminomas and dysgerminoma were negative, non-seminoma cells were frequently found to express beta-catenin. Expression of both factors therefore reflects the degree of differentiation of these tumours. No differences for either E-cadherin or beta-catenin were observed between samples of tumours resistant or sensitive to chemotherapy, and E-cadherin expression did not correlate with vascular invasion. E-cadherin and beta-catenin therefore play a role in both normal and malignant germ cell development and differentiation that warrants further investigation, but they seem to be of limited value as predictive or prognostic factors in GCTs.

Although our results are in line with previous findings of the presence of wild-type P53 in TGCTs, they show that a high level of P53 does not relate directly to treatment sensitivity of these tumors, and inactivation of P53 is not a common event in the development of cisplatin resistance.