Pnck induces ligand-independent EGFR degradation by probable perturbation of the Hsp90 chaperone complex

We have recently described a novel role for pregnancy-upregulated nonubiquitous calmodulin kinase (Pnck) in the induction of ligand-independent epidermal growth factor receptor (EGFR) degradation (Deb TB, Coticchia CM, Barndt R, Zuo H, Dickson RB, and Johnson MD. Am J Physiol Cell Physiol 295: C365–C377, 2008). In the current communication, we explore the probable mechanism by which Pnck induces ligand-independent EGFR degradation. Pnck-induced EGFR degradation is calcium/calmodulin independent and is regulated by cell density, with the highest EGFR degradation observed at low cell density. Pnck is a novel heat shock protein 90 (Hsp90) client protein that can be coimmunoprecipitated with Hsp90. Treatment of Pnck-overexpressing cells with the pharmacologic Hsp90 inhibitor geldanamycin results in enhanced EGFR degradation, and destruction of Pnck. In cells in which Pnck is inducing EGFR degradation, we observed that Hsp90 exhibits reduced electrophoretic mobility, and through mass spectrometric analysis of immunopurified Hsp90 protein we demonstrated enhanced phosphorylation at threonine 89 and 616 (in both Hsp90-α and -β) and serine 391 (in Hsp90-α). Kinase-active Pnck protein is degraded by the proteasome, concurrent with EGFR degradation. A Pnck mutant (T171A) protein with suppressed kinase activity induced EGFR degradation to essentially the same level as wild-type (WT) Pnck, suggesting that Pnck kinase activity is not required for the induction of EGFR degradation. Although EGFR is degraded, overexpression of WT Pnck paradoxically promoted cellular proliferation, whereas cells expressing mutant Pnck (T171A) were growth inhibited. WT Pnck promoted S to G2 transition, but cells expressing the mutant exhibited higher residency time in S phase. Basal MAP kinase activity was inhibited by WT Pnck but not by mutant T171A Pnck protein. Cyclin-dependent kinase (Cdk) inhibitor p21/Cip-1/Waf-1 was transcriptionally suppressed downstream to MAP kinase inhibition by WT Pnck, but not the mutant protein. Collectively, these data suggest that 1) Pnck induces ligand-independent EGFR degradation most likely through perturbation of Hsp90 chaperone activity due to Hsp90 phosphorylation, 2) EGFR degradation is coupled to proteasomal degradation of Pnck, and 3) modulation of basal MAP kinase activity, p21/Cip-1/Waf-1 expression, and cellular growth by Pnck is independent of Pnck-induced ligand-independent EGFR degradation.

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Differential Activation of Mitogen-Activated Protein Kinases in Experimental Mesangioproliferative Glomerulonephritis

Journal of the American Society of Nephrology ◽

10.1681/asn.v112232 ◽

2000 ◽

Vol 11 (2) ◽

pp. 232-240

Author(s):

DIRK BOKEMEYER ◽

TAMMO OSTENDORF ◽

UTA KUNTER ◽

MARION LINDEMANN ◽

HERBERT J. KRAMER ◽

...

Keyword(s):

Map Kinase ◽

Map Kinases ◽

Cellular Proliferation ◽

Kinase Activity ◽

P38 Map Kinase ◽

Cellular Growth ◽

Terminal Deoxynucleotidyl Transferase ◽

Maximal Increase

Abstract. Multiple extracellular mitogens are involved in the pathogenesis of proliferative forms of glomerulonephritis (GN). In vitro studies demonstrate the pivotal role of mitogenactivated protein (MAP) kinases in the regulation of cellular proliferation. This study was conducted to examine whether these kinases, as a convergence point of mitogenic stimuli, are activated in mesangioproliferative GN in vivo. Therefore, anti-Thy1 GN was induced in rats using a monoclonal anti-Thy1.1 antibody (OX-7). Whole cortical tissue as well as isolated glomeruli were examined at different time points using kinase activity assays and Western blot analysis. A maximal increase in the number of glomerular mitotic figures (9.7-fold) was demonstrated 6 d after injection of the anti-Thy1.1 antibody. In parallel with this finding, a significant increase in cortical, and more dramatically glomerular, activity of extracellular signal-regulated kinase (ERK) was detected. Maximal activation of ERK was detectable on day 6. This activation of ERK was accompanied by an increase in the expression of MEK (MAP kinase/ERK kinase), the ERK-activating kinase. A marked induction of glomerular apoptosis at 2 h after injection of the anti-Thy1.1 antibody, which subsided subsequently, was demonstrated using the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay as well as staining for single-stranded DNA. However, no significant activation of stress-activated protein kinase or p38 MAP kinase, both MAP kinases that are suggested to induce apoptosis and to inhibit cellular growth, was detectable at this early time point. Rather, on day 6 a dramatic decrease in the activity of p38 MAP kinase, which might have contributed to the overshooting glomerular cellular proliferation, was observed. Treatment of rats with heparin blunted glomerular proliferation as well as ERK activation and restored p38 MAP kinase activity. These observations point to ERK and p38 MAP kinase as putative mediators of the proliferative response in mesangioproliferative GN and suggest that upregulation of MEK is involved in the long-term regulation of ERK in vivo.

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Inhibition of c-kit receptor tyrosine kinase activity by STI 571, a selective tyrosine kinase inhibitor

Blood ◽

10.1182/blood.v96.3.925 ◽

2000 ◽

Vol 96 (3) ◽

pp. 925-932 ◽

Cited By ~ 729

Author(s):

Michael C. Heinrich ◽

Diana J. Griffith ◽

Brian J. Druker ◽

Cecily L. Wait ◽

Kristen A. Ott ◽

...

Keyword(s):

Tyrosine Kinase ◽

Map Kinase ◽

Cell Line ◽

Cellular Proliferation ◽

Kinase Activity ◽

Leukemia Cell ◽

Myelogenous Leukemia ◽

Leukemia Cell Line ◽

Tyrosine Kinase Activity ◽

Sti 571

Abstract STI 571 (formerly known as CGP 57148B) is a known inhibitor of the c-abl, bcr-abl, and platelet-derived growth-factor receptor (PDGFR) tyrosine kinases. This compound is being evaluated in clinical trials for the treatment of chronic myelogenous leukemia. We sought to extend the activity profile of STI 571 by testing its ability to inhibit the tyrosine kinase activity of c-kit, a receptor structurally similar to PDGFR. We treated a c-kit expressing a human myeloid leukemia cell line, M-07e, with STI 571 before stimulation with Steel factor (SLF). STI 571 inhibited c-kit autophosphorylation, activation of mitogen-activated protein (MAP) kinase, and activation of Akt without altering total protein levels of c-kit, MAP kinase, or Akt. The concentration that produced 50% inhibition for these effects was approximately 100 nmol/L. STI 571 also significantly decreased SLF-dependent growth of M-07e cells in a dose-dependent manner and blocked the antiapoptotic activity of SLF. In contrast, the compound had no effect on MAP kinase activation or cellular proliferation in response to granulocyte-macrophage colony-stimulating factor. We also tested the activity of STI 571 in a human mast cell leukemia cell line (HMC-1), which has an activated mutant form of c-kit. STI 571 had a more potent inhibitory effect on the kinase activity of this mutant receptor than it did on ligand-dependent activation of the wild-type receptor. These findings show that STI 571 selectively inhibits c-kit tyrosine kinase activity and downstream activation of target proteins involved in cellular proliferation and survival. This compound may be useful in treating cancers associated with increased c-kit kinase activity.

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Inhibition of c-kit receptor tyrosine kinase activity by STI 571, a selective tyrosine kinase inhibitor

Blood ◽

10.1182/blood.v96.3.925.015k50_925_932 ◽

2000 ◽

Vol 96 (3) ◽

pp. 925-932 ◽

Cited By ~ 15

Author(s):

Michael C. Heinrich ◽

Diana J. Griffith ◽

Brian J. Druker ◽

Cecily L. Wait ◽

Kristen A. Ott ◽

...

Keyword(s):

Tyrosine Kinase ◽

Map Kinase ◽

Cell Line ◽

Cellular Proliferation ◽

Kinase Activity ◽

Leukemia Cell ◽

Myelogenous Leukemia ◽

Leukemia Cell Line ◽

Tyrosine Kinase Activity ◽

Sti 571

STI 571 (formerly known as CGP 57148B) is a known inhibitor of the c-abl, bcr-abl, and platelet-derived growth-factor receptor (PDGFR) tyrosine kinases. This compound is being evaluated in clinical trials for the treatment of chronic myelogenous leukemia. We sought to extend the activity profile of STI 571 by testing its ability to inhibit the tyrosine kinase activity of c-kit, a receptor structurally similar to PDGFR. We treated a c-kit expressing a human myeloid leukemia cell line, M-07e, with STI 571 before stimulation with Steel factor (SLF). STI 571 inhibited c-kit autophosphorylation, activation of mitogen-activated protein (MAP) kinase, and activation of Akt without altering total protein levels of c-kit, MAP kinase, or Akt. The concentration that produced 50% inhibition for these effects was approximately 100 nmol/L. STI 571 also significantly decreased SLF-dependent growth of M-07e cells in a dose-dependent manner and blocked the antiapoptotic activity of SLF. In contrast, the compound had no effect on MAP kinase activation or cellular proliferation in response to granulocyte-macrophage colony-stimulating factor. We also tested the activity of STI 571 in a human mast cell leukemia cell line (HMC-1), which has an activated mutant form of c-kit. STI 571 had a more potent inhibitory effect on the kinase activity of this mutant receptor than it did on ligand-dependent activation of the wild-type receptor. These findings show that STI 571 selectively inhibits c-kit tyrosine kinase activity and downstream activation of target proteins involved in cellular proliferation and survival. This compound may be useful in treating cancers associated with increased c-kit kinase activity.

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HSP90 inhibitor NVP-BEP800 affects stability of SRC kinases and growth of T-cell and B-cell acute lymphoblastic leukemias

Blood Cancer Journal ◽

10.1038/s41408-021-00450-2 ◽

2021 ◽

Vol 11 (3) ◽

Author(s):

Rony Mshaik ◽

John Simonet ◽

Aleksandra Georgievski ◽

Layla Jamal ◽

Shaliha Bechoua ◽

...

Keyword(s):

T Cell ◽

B Cell ◽

Treatment Strategies ◽

Heat Shock Protein 90 ◽

Competitive Inhibitor ◽

Hsp90 Inhibitor ◽

Src Kinases ◽

Risk Patients ◽

Hsp90 Chaperone ◽

New Treatment

AbstractT-cell and B-cell acute lymphoblastic leukemias (T-ALL, B-ALL) are aggressive hematological malignancies characterized by an accumulation of immature T- or B-cells. Although patient outcomes have improved, novel targeted therapies are needed to reduce the intensity of chemotherapy and improve the prognosis of high-risk patients. Using cell lines, primary cells and patient-derived xenograft (PDX) models, we demonstrate that ALL cells viability is sensitive to NVP-BEP800, an ATP-competitive inhibitor of Heat shock protein 90 (HSP90). Furthermore, we reveal that lymphocyte-specific SRC family kinases (SFK) are important clients of the HSP90 chaperone in ALL. When PDX mice are treated with NVP-BEP800, we found that there is a decrease in ALL progression. Together, these results demonstrate that the chaperoning of SFK by HSP90 is involved in the growth of ALL. These novel findings provide an alternative approach to target SRC kinases and could be used for the development of new treatment strategies for ALL.

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Heat Shock Protein 90 Chaperones E1A Early Protein of Adenovirus 5 and Is Essential for Replication of the Virus

International Journal of Molecular Sciences ◽

10.3390/ijms22042020 ◽

2021 ◽

Vol 22 (4) ◽

pp. 2020

Author(s):

Iga Dalidowska ◽

Olga Gazi ◽

Dorota Sulejczak ◽

Maciej Przybylski ◽

Pawel Bieganowski

Keyword(s):

Mrna Level ◽

Heat Shock Protein 90 ◽

Human Adenovirus ◽

Hsp90 Inhibitor ◽

Hsp90 Inhibition ◽

Early Protein ◽

Fold Reduction ◽

Hsp90 Chaperone ◽

Adenovirus 5 ◽

E1a Protein

Adenovirus infections tend to be mild, but they may pose a serious threat for young and immunocompromised individuals. The treatment is complicated because there are no approved safe and specific drugs for adenovirus infections. Here, we present evidence that 17-(Allylamino)-17-demethoxygeldanamycin (17-AAG), an inhibitor of Hsp90 chaperone, decreases the rate of human adenovirus 5 (HAdV-5) replication in cell cultures by 95%. 17-AAG inhibited the transcription of early and late genes of HAdV-5, replication of viral DNA, and expression of viral proteins. 6 h after infection, Hsp90 inhibition results in a 6.3-fold reduction of the newly synthesized E1A protein level without a decrease in the E1A mRNA level. However, the Hsp90 inhibition does not increase the decay rate of the E1A protein that was constitutively expressed in the cell before exposure to the inhibitor. The co-immunoprecipitation proved that E1A protein interacted with Hsp90. Altogether, the presented results show, for the first time. that Hsp90 chaperones newly synthesized, but not mature, E1A protein. Because E1A serves as a transcriptional co-activator of adenovirus early genes, the anti-adenoviral activity of the Hsp90 inhibitor might be explained by the decreased E1A level.

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