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1.
Mol Cancer Ther ; 18(5): 937-946, 2019 05.
Article in English | MEDLINE | ID: mdl-30926642

ABSTRACT

Protein tyrosine kinase 6 (PTK6, also called BRK) is overexpressed and activated in human prostate cancer. Loss of the tumor suppressor PTEN, a frequent event in prostate cancer, leads to PTK6 activation at the plasma membrane and its oncogenic signaling. The small molecule inhibitor vemurafenib, also known as PLX4032, and its tool analog PLX4720 were designed to inhibit constitutively active BRAF V600E, yet they also have potent effects against PTK6. Vemurafenib is used in the treatment of metastatic melanoma, but its efficacy in prostate cancer has not been assessed. When activated at the plasma membrane, PTK6 promotes signaling through FAK, EGFR, and ERK1/2, and we show this can be blocked by vemurafenib. In addition, PTK6-mediated cell growth, migration, and invasion are inhibited upon vemurafenib administration. Using a flank xenograft model, vemurafenib treatment reduced tumor burden. Using saturation transfer difference NMR and molecular docking, we demonstrate that vemurafenib binds in the active site of PTK6, inhibiting its activation. These structural studies provide insight into the PTK6-vemurafenib complex, which can be utilized for further refinement chemistry, whereas functional studies demonstrate that active PTK6 is a viable drug target in prostate cancer.


Subject(s)
Neoplasm Proteins/chemistry , Prostatic Neoplasms/drug therapy , Protein-Tyrosine Kinases/chemistry , Vemurafenib/pharmacology , Animals , Cell Movement/drug effects , Cell Proliferation/drug effects , ErbB Receptors/genetics , Focal Adhesion Kinase 1/genetics , Heterografts , Humans , MAP Kinase Signaling System/drug effects , Male , Mice , Molecular Docking Simulation , Neoplasm Invasiveness/genetics , Neoplasm Invasiveness/pathology , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/genetics , PTEN Phosphohydrolase/genetics , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology , Protein-Tyrosine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/genetics , Proto-Oncogene Proteins B-raf/genetics , Signal Transduction/drug effects , Vemurafenib/chemistry
2.
Nat Commun ; 8(1): 1508, 2017 11 15.
Article in English | MEDLINE | ID: mdl-29142193

ABSTRACT

PTEN activity is often lost in prostate cancer. We show that the tyrosine kinase PTK6 (BRK) is a PTEN substrate. Phosphorylation of PTK6 tyrosine 342 (PY342) promotes activation, while phosphorylation of tyrosine 447 (PY447) regulates auto-inhibition. Introduction of PTEN into a PTEN null prostate cancer cell line leads to dephosphorylation of PY342 but not PY447 and PTK6 inhibition. Conversely, PTEN knockdown promotes PTK6 activation in PTEN positive cells. Using a variety of PTEN mutant constructs, we show that protein phosphatase activity of PTEN targets PTK6, with efficiency similar to PTP1B, a phosphatase that directly dephosphorylates PTK6 Y342. Conditional disruption of Pten in the mouse prostate leads to tumorigenesis and increased phosphorylation of PTK6 Y342, and disruption of Ptk6 impairs tumorigenesis. In human prostate tumor tissue microarrays, loss of PTEN correlates with increased PTK6 PY342 and poor outcome. These data suggest PTK6 activation promotes invasive prostate cancer induced by PTEN loss.


Subject(s)
Neoplasm Proteins/metabolism , PTEN Phosphohydrolase/metabolism , Protein-Tyrosine Kinases/metabolism , Signal Transduction , Animals , Cell Line, Tumor , Gene Expression Regulation, Neoplastic , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mutation , Neoplasm Proteins/genetics , PTEN Phosphohydrolase/genetics , Phosphorylation , Protein-Tyrosine Kinases/genetics , Tissue Array Analysis
3.
Microbiology (Reading) ; 158(Pt 10): 2568-2576, 2012 Oct.
Article in English | MEDLINE | ID: mdl-22902728

ABSTRACT

The clpC operon in Staphylococcus aureus comprises four genes, denoted ctsR, mcsA, mcsB and clpC. A mutation within the mcsB gene resulted in hypersensitivity to heavy metal stress, temperature stress, osmotic pressure stress and oxidative stress. This mutation also resulted in sensitivity to variations in pH and lowered expression of the clpC operon under adverse extracellular conditions, as determined by quantitative real-time PCR (qRT-PCR). Additionally, virulence traits such as haemolytic activity, proteolysis, biofilm formation, and evasion from peritoneal fluid killing were substantially reduced in the ΔmcsB strain. Interestingly, mutated mcsB also caused a significant reduction in expression of virulence determinants hla and saeS. To be a successful pathogen, S. aureus must effectively overcome these types of stresses that are encountered within the host. These data show that an S. aureus strain lacking functional mcsB is stress hypersensitive and therefore less viable when introduced into hostile environments. For the first time, these studies have identified mcsB as a crucial and necessary component of stress and pathogenicity mechanisms.


Subject(s)
Bacterial Proteins/genetics , Genes, Bacterial , Heat-Shock Proteins/genetics , Heat-Shock Response , Operon , Phosphotransferases/metabolism , Staphylococcus aureus/physiology , Staphylococcus aureus/pathogenicity , Bacterial Proteins/metabolism , Gene Expression Regulation, Bacterial , Heat-Shock Proteins/metabolism , Humans , Mutation , Phosphotransferases/genetics , Staphylococcus aureus/genetics , Staphylococcus aureus/metabolism , Virulence/genetics
4.
Mob Genet Elements ; 1(1): 8-17, 2011 May.
Article in English | MEDLINE | ID: mdl-22016841

ABSTRACT

MicroRNAs (miRs) are small non-coding RNAs that generally function as negative regulators of target messenger RNAs (mRNAs) at the posttranscriptional level. MiRs bind to the 3'UTR of target mRNAs through complementary base pairing, resulting in target mRNA cleavage or translation repression. To date, over 15,000 distinct miRs have been identified in organisms ranging from viruses to man and interest in miR research continues to intensify. Of note, the most enlightening aspect of miR function-the mRNAs they target-continues to be elusive. Descriptions of the molecular origins of independent miR molecules currently support the hypothesis that miR hairpin generation is based on the adjacent insertion of two related transposable elements (TEs) at one genomic locus. Thus transcription across such TE interfaces establishes many, if not the majority of functional miRs. The implications of these findings are substantial for understanding how TEs confer increased genomic fitness, describing miR transcriptional regulations and making accurate miR target predictions. In this work, we have performed a comprehensive analysis of the genomic events responsible for the formation of all currently annotated miR loci. We find that the connection between miRs and transposable elements is more significant than previously appreciated, and more broadly, supports an important role for repetitive elements in miR origin, expression and regulatory network formation. Further, we demonstrate the utility of these findings in miR target prediction. Our results greatly expand the existing repertoire of defined miR origins, detailing the formation of 2,392 of 15,176 currently recognized miR genomic loci and supporting a mobile genetic element model for the genomic establishment of functional miRs.

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