Search

Hyperactivation of SRC-family protein kinases (SFKs) contributes to the initiation and progression of human colorectal cancer (CRC). Since oncogenic mutations of SFKgenes are rare in human CRC, we investigated if SFK hyperactivation is linked to dysregulation of their upstream inhibitors, C-terminal SRC kinase (CSK) and its homolog CSK-homologous kinase (CHK/MATK). We demonstrate that expression of CHK/MATKbut not CSKwas significantly downregulated in CRC cell lines and primary tumours compared to normal colonic tissue. Investigation of the mechanism by which CHK/MATKexpression is down-regulated in CRC cells uncovered hypermethylation of the CHK/MATKpromoter in CRC cell lines and primary tumours. Promoter methylation of CHK/MATKwas also observed in several other tumour types. Consistent with epigenetic silencing of CHK/MATK, genetic deletion or pharmacological inhibition of DNA methyltransferases increased CHK/MATKmRNA expression in CHK/MATK-methylated colon cancer cell lines. SFKs were hyperactivated in CHK/MATK-methylated CRC cells despite expressing enzymatically active CSK, suggesting loss of CHK/MATK contributes to SFK hyperactivation. Re-expression of CHK/MATK in CRC cell lines led to reduction in SFK activity via a non-catalytic mechanism, a reduction in anchorage-independent growth, cell proliferation and migration in vitro, and a reduction in tumour growth and metastasis in a zebrafish embryo xenotransplantation model in vivo, collectively identifying CHK/MATKas a novel putative tumour suppressor gene in CRC. Furthermore, our discovery that CHK/MATKhypermethylation occurs in the majority of tumours warrants its further investigation as a diagnostic marker of CRC.

The von Hippel–Lindau tumor suppressor protein (VHL), when mutated and inactivated, has been associated with renal and CNS cancer development. VHL normally plays an important role in targeting for degradation of the HIF-1a (hypoxia inducible factor-1a) transcription factor, a primary positive regulator of vascular endothelial growth factor (VEGF) production. In this report we demonstrate that VHL destabilization can be induced by Src kinase and may be involved in other cancers, including breast cancer. We have found that elevated Src can trigger a drastic reduction in VHL stability even under normoxic conditions, through phosphorylation of VHL tyrosine residue 185, leading to ubiquitination and proteasome-mediated degradation of VHL. The Src-induced degradation of VHL protein leads to increased HIF-1a levels and transcriptional activity and increased VEGF production. In this manner, Src regulation of VHL protein stability may play an important role in promoting VEGF expression, tumor angiogenesis, and cancer progression.

Two activated transforming mutants of human pp60c-srcwere found to possess single point mutations within the regulatory carboxyl terminus (E527K in CY CST201) and the kinase domain (E381G in WO CST1), respectively, that do not directly interfere with either the regulatory c-Src kinase (CSK) phosphorylation site (Tyr530) or the SH2/3 domains. In vivo, both mutant proteins are hypophosphorylated on their carboxyl-terminal regulatory tyrosines and are hyperactive. In an in vitroSrc kinase inactivation assay, both mutant Src proteins exhibited resistance to inactivation by CSK relative to wild-type Src. Under these in vitroconditions, E381G c-Src was found to be phosphorylated by CSK to wild-type levels, while E527K c-Src was not detectably phosphorylated. The ability of CSK to phosphorylate a carboxyl-terminal peptide modelled against E527K c-Src was also impaired, suggesting that CSK is unable to recognize E527K c-Src as an efficient substrate. In the case of E381G c-Src, examination of whether its SH2/3 domains were accessible to the carboxyl-terminal regulatory phosphotyrosine revealed a highly reduced ability of autophosphorylated E381G c-Src to bind to a synthetic phosphopeptide modelled from the SH2-binding region of polyoma middle-T antigen which binds to Src SH2 with high affinity. This suggests that the E381G c-Src mutation results in an altered or reduced accessibility of the SH2 domain of the autophosphorylated form of E381G c-Src and may represent a previously undescribed mode of Src activation. Further study of these and other Src mutants may offer additional new insights into the regulation of “Src family” kinases.

The envgene of avian sarcoma-leukosis viruses codes for envelope glycoproteins that determine viral host range, antigenic specificity, and interference patterns. We used molecular hybridization to analyze the natural distribution and possible origins of the nucleotide sequences that encode env; our work exploited the availability of radioactive DNA (cDNAgp) complementary to most or all of env. envsequences were detectable in the DNAs of chickens which synthesized an envgene product (chick helper factor positive) encoded by an endogenous viral gene and also in the DNAs of chickens which synthesized little or no envgene product (chick helper factor negative). envsequences were not detectable in DNAs from Japanese quail, ring-necked pheasant, golden pheasant, duck, squab, salmon sperm, or calf thymus. The detection of sequences closely related to viral envonly in chicken DNA contrasts sharply with the demonstration that the transforming gene (src) of avian sarcoma viruses has readily detectable homologues in the DNAs of all avian species tested [D. Stehelin, H. E. Varmus, J. M. Bishop, and P. K. Vogt, Nature (London) 260: 170-173, 1976] and in the DNAs of other vertebrates (D. Spector, personal communication). Thermal denaturation studies on duplexes formed between cDNAgpand chicken DNA and also between cDNAgpand RNAs of subgroup A to E viruses derived from chickens indicated that these duplexes were well matched. In contrast, cDNAgpdid not form stable hybrids with RNAs of viruses which were isolated from ring-necked and golden pheasants. We conclude that substantial portions of nucleotide sequences within the envgenes of viruses of subgroups A to E are closely related and that these genes probably have a common, perhaps cellular, evolutionary origin.

The cytoplasmic protein tyrosine kinase p56lckhas been implicated as an effector of interleukin-2-induced cell division in T-lymphocytes, but little is known about physiological substrates for p56lckduring these events. We have used p56lckfusion proteins to identify potential cytoplasmic signal transduction proteins that bind to p56lckin mitotically activated human peripheral blood lymphocytes and in constitutively dividing leukemic T-cell lines. In peripheral blood lymphocytes, we have observed an interleukin-2-dependent tyrosine phosphorylation of a 70-kDa protein and binding of tyrosine phosphorylated p70 to the SH2 domain of p56lck. A 70-kDa phosphoprotein was also observed to constitutively bind p56lckin leukemic T-cells. Affinity purification of p56lck-associated p70 and sequencing of proteolytic fragments revealed identity to a 62-kDa protein that has been identified as a ras-GTPase activating protein. These results demonstrate a stimulation-dependent tyrosine phosphorylation of p70 and its interaction with p56lckand may provide a link between p56lckand GTPase-mediated signal transduction pathways in activated T-lymphocytes.

The effect of nalidixic acid on deoxyribonucleic acid (DNA) synthesis in Bacillus subtiliscells infected with bacteriophage SPO1 was studied. Nalidixic acid had little inhibitory effect on SPO1 DNA synthesis at concentrations that drastically inhibited B. subtilisDNA synthesis. Inhibition of DNA synthesis, appropriate to the concentration used, was imposed within 1 min after addition of nalidixic acid, suggesting that it acts directly on DNA synthesis in both infected and uninfected cells. The SPO1 DNA synthesized in the presence of high concentrations of nalidixic acid had a density characteristic of normal SPO1 DNA and was packaged into viable progeny phage particles, but its rate of synthesis was reduced and bacterial lysis was delayed.