Inhibition of Hsp90 via 17-DMAG induces apoptosis in a p53-dependent manner to prevent medulloblastoma.

Elevated expression of HSP90 is observed in many tumor types and is associated with a limited clinical response. Targeting HSP90 using inhibitors such as 17-DMAG (17-desmethoxy-17-N,N-dimethylaminoethylaminogeldanamycin) has shown limited therapeutic success. HSP90 regulates the function of several proteins implicated in tumorigenesis although the precise mechanism through which 17-DMAG regulates tumor cell survival remains unclear. We observed a requirement for p53 in mediating 17-DMAG-induced cell death. The sensitivity of primary mouse embryonic fibroblasts and tumor cells to 17-DMAG-induced apoptosis depended on the p53 status. Wild-type MEFs underwent 17-DMAG-induced caspase-dependent cell death, whilst those lacking p53 failed to do so. Interestingly p53-dependent cell death occurred independently of Atm or Arf. Primary tumor cells derived from two models of murine medulloblastoma (Ptch1(+/-);Ink4c(-/-) and p53(FL/FL);Nestin-Cre(+); Ink4c(-/-)) that retain and lack p53 function, respectively, displayed a dependence on functional p53 to engage 17-DMAG-induced apoptosis. Strikingly, 17-DMAG treatment in an allograft model of Ptch1(+/-);Ink4c(-/-) but not p53(FL/FL);Nestin-Cre(+); Ink4c(-/-) tumor cells prevented tumor growth in vivo. Our data suggest that p53 status is a likely predictor of the sensitivity of tumors to 17-DMAG.

Jak2 tyrosine kinase and cancer: how good cells get HiJAKed.

Cloned in 1992, Jak2 tyrosine kinase has emerged as a critical molecule in mammalian development, physiology, and disease. Here, we will review the early history of Jak2 as it pertains to its role in classical cellular signaling. We also review how specific structural determinants within Jak2 dictate its overall function. Finally, we will review relatively recent literature as it pertains to the role of Jak2 in neoplastic growth as well as the identification of novel Jak2 inhibitors. It is our hope that by reviewing these specific areas, we will have a better understanding of the role of Jak2 in cancer, and in turn, we may have a better idea as to how to block aberrant Jak2 function.

The N-terminal SH2 domain of the tyrosine phosphatase, SHP-2, is essential for Jak2-dependent signaling via the angiotensin II type AT1 receptor.

Previous work has suggested that the protein tyrosine phosphatase, SHP-2, may act to facilitate angiotensin II (Ang II)-mediated, Jak2-dependent signaling. However, the mechanisms by which this occurs are not known. Here, Ang II-mediated, Jak2-dependent signaling was analyzed in a fibroblast cell line lacking the N-terminal, SH2 domain of SHP-2 (SHP-2(Delta46-110)). While the SHP-2(Delta46-110) cells were capable of activating Jak2 tyrosine kinase, they were unable to facilitate AT1 receptor/Jak2 co-association, STAT activation and subsequent Ang II-mediated gene transcription when compared to wild type control cells. These data therefore suggested that the N-terminal SH2 domain of SHP-2 was acting to recruit Jak2 to the AT1 receptor signaling complex. We found that the N-terminal SH2 domain of SHP-2 binds Jak2 predominantly, but not exclusively at tyrosine 201. Mass spectrometry analysis confirmed that this tyrosine residue is in fact phosphorylated. When this tyrosine was converted to phenylalanine, the ability of Jak2 to activate subsequent downstream signaling events was reduced. In summary, we have identified a novel site of Jak2 tyrosine autophosphorylation; namely, tyrosine 201. Our data suggest that the N-terminal SH2 domain of SHP-2 binds this amino acid residue. The functional consequence of this interaction is to recruit Jak2 to the AT1 receptor signaling complex and in turn promote downstream Jak2-dependent signaling.

ERK1/2 regulates ANG II-dependent cell proliferation via cytoplasmic activation of RSK2 and nuclear activation of elk1.

In a concurrently submitted article, we show that ANG II-induced ERK1/2 activation is mediated by both c-Src/Yes/Fyn and heterotrimeric G protein/PKCzeta-dependent signaling. Furthermore, we show that heterotrimeric G protein/PKCzeta-activated ERK1/2 is destined for the nucleus while ERK1/2 activated by c-Src/Yes/Fyn-dependent signaling remains in the cytoplasm. Interestingly, both mechanisms of activation are required for maximum ANG II-induced cell proliferation. In this study, we sought to determine the mechanisms by which ERK1/2 facilitate cell proliferation via these distinct nuclear and cytoplasmic events, using cells that were lacking either c-Src/Yes/Fyn or heterotrimeric G protein/PKCzeta-dependent ERK1/2 activation. A loss of c-Src/Yes/Fyn blocked ANG II-dependent RSK2 activation, RSK2 nuclear translocation, serum-response factor (SRF) phosphorylation, a portion of c-fos transcriptional activity and c-Fos phosphorylation. Blocking ANG II-induced heterotrimeric G protein/PKCzeta activity resulted in a loss of ERK1/2 nuclear translocation, elk1 phosphorylation, and the remaining portion of c-fos transcriptional activity not dependent on c-Src/Yes/Fyn. Inhibition of RSK with the potent and selective inhibitor, SL0101, attenuated ANG II-induced cell proliferation, and, in combination with a PKCzeta pseudosubstrate, completely attenuated cell proliferation. Thus we conclude that ERK1/2 mediate ANG II-dependent cell proliferation via distinct cytoplasmic and nuclear signaling events, which are in turn governed by c-Src/Yes/Fyn and heterotrimeric G protein/PKCzeta-dependent signaling, respectively.

ANG II-induced cell proliferation is dually mediated by c-Src/Yes/Fyn-regulated ERK1/2 activation in the cytoplasm and PKCzeta-controlled ERK1/2 activity within the nucleus.

High-affinity binding of angiotensin II (ANG II) to the ANG II type 1 receptor (AT(1)R) results in the activation of ERK1/2 mitogen-activated protein kinases (MAPK). However, the precise mechanism of ANG II-induced ERK1/2 activation has not been fully characterized. Here, we investigated the signaling events leading to ANG II-induced ERK1/2 activation using a c-Src/Yes/Fyn tyrosine kinase-deficient mouse embryonic fibroblast (MEF) cell line stably transfected with the AT(1)R (SYF/AT(1)). ERK1/2 activation was reduced by approximately 50% within these cells compared with wild-type controls (WT/AT(1)). The remaining approximately 50% of intracellular ERK1/2 activation was dependent upon heterotrimeric G protein and protein kinase C zeta (PKCzeta) activation. Therefore, ANG II-induced ERK1/2 activation occurs via two independent mechanisms. We next investigated whether a loss of either c-Src/Yes/Fyn or PKCzeta signaling affected ERK1/2 nuclear translocation and cell proliferation in response to ANG II. ANG II-induced cell proliferation was markedly reduced in SYF/AT(1) cells compared with WT/AT(1) cells (P < 0.01), but interestingly, ERK2 nuclear translocation was normal. ANG II-induced nuclear translocation of ERK2 was blocked via pretreatment of WT/AT(1) cells with a PKCzeta pseudosubstrate. ANG II-induced cell proliferation was significantly reduced in PKCzeta pseudosubstrate-treated WT/AT(1) cells (P < 0.01) and was completely blocked in SYF/AT(1) cells treated with this same compound. Thus ANG II-induced cell proliferation appears to be regulated by both ERK1/2-driven nuclear and cytoplasmic events. In response to ANG II, the ability of ERK1/2 to remain within the cytoplasm or translocate into the nucleus is controlled by c-Src/Yes/Fyn or heterotrimeric G protein/PKCzeta signaling, respectively.

Jak2 tyrosine kinase: a true jak of all trades?

Discovered roughly 10 yr ago, Jak2 tyrosine kinase has emerged as a critical molecule in mammalian development, physiology, and disease. Here, we review the early history of Jak2 and its role in health and disease. We will also review its critical role in mediating cytokine-dependent signal transduction. Additionally, more recent work demonstrating the importance of Jak2 in G protein-coupled receptor and tyrosine kinase growth factor receptor signal transduction will be discussed. The cellular and biochemical mechanisms by which Jak2 tyrosine kinase is activated and regulated within the cell also will be reviewed. Finally, structure-function and pharmacological-based studies that identified structural motifs and amino acids within Jak2 that are critical for its function will be examined. By reviewing the biology of Jak2 tyrosine kinase at the molecular, cellular, and physiological levels, we hope to advance the understanding of how a single gene can have such a profound impact on development, physiology, and disease.

Jak2 tyrosine kinase mediates angiotensin II-dependent inactivation of ERK2 via induction of mitogen-activated protein kinase phosphatase 1.

Previous work has shown that inhibition of Jak2 via the pharmacological compound AG490 blocks the angiotensin II (Ang II)-dependent activation of ERK2, thereby suggesting an essential role of Jak2 in ERK activation. However, recent studies have thrown into question the specificity of AG490 and therefore the role of Jak2 in ERK activation. To address this, we reconstituted an Ang II signaling system in a Jak2-/-cell line and measured the ability of Ang II to activate ERK2 in these cells. Controls for this study were the same cells expressing Jak2 via the addition of a Jak2 expression plasmid. In the cells expressing Jak2, Ang II induced a marked increase in ERK2 activity as measured by Western blot analysis and in vitro kinase assays. ERK2 activity returned to basal levels within 30 min. However, in the cells lacking Jak2, Ang II treatment resulted in ERK2 activation that did not return to basal levels until 120 min after ligand addition. Analysis of phosphatase gene expression revealed that Ang II induced mitogen-activated protein kinase phosphatase 1 (MKP-1) expression in cells expressing Jak2 but failed to induce MKP-1 expression in cells lacking Jak2. Therefore, our results suggest that Jak2 is not required for Ang II-induced ERK2 activation. Rather Jak2 is required for Ang II-induced ERK2 inactivation via induction of MKP-1 gene expression.