title: Targeting of nucleotide-binding proteins by HAMLET--a conserved tumor cell death mechanism.
abstract: HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) kills tumor cells broadly suggesting that conserved survival pathways are perturbed. We now identify nucleotide-binding proteins as HAMLET binding partners, accounting for about 35% of all HAMLET targets in a protein microarray comprising 8000 human proteins. Target kinases were present in all branches of the Kinome tree, including 26 tyrosine kinases, 10 tyrosine kinase-like kinases, 13 homologs of yeast sterile kinases, 4 casein kinase 1 kinases, 15 containing PKA, PKG, PKC family kinases, 15 calcium/calmodulin-dependent protein kinase kinases and 13 kinases from CDK, MAPK, GSK3, CLK families. HAMLET acted as a broad kinase inhibitor in vitro, as defined in a screen of 347 wild-type, 93 mutant, 19 atypical and 17 lipid kinases. Inhibition of phosphorylation was also detected in extracts from HAMLET-treated lung carcinoma cells. In addition, HAMLET recognized 24 Ras family proteins and bound to Ras, RasL11B and Rap1B on the cytoplasmic face of the plasma membrane. Direct cellular interactions between HAMLET and activated Ras family members including Braf were confirmed by co-immunoprecipitation. As a consequence, oncogenic Ras and Braf activity was inhibited and HAMLET and Braf inhibitors synergistically increased tumor cell death in response to HAMLET. Unlike most small molecule kinase inhibitors, HAMLET showed selectivity for tumor cells in vitro and in vivo. The results identify nucleotide-binding proteins as HAMLET targets and suggest that dysregulation of the ATPase/kinase/GTPase machinery contributes to cell death, following the initial, selective recognition of HAMLET by tumor cells. The findings thus provide a molecular basis for the conserved tumoricidal effect of HAMLET, through dysregulation of kinases and oncogenic GTPases, to which tumor cells are addicted.
authors.affiliation: Department of Microbiology, Immunology and Glycobiology (MIG), Institute of Laboratory Medicine, Lund University, Lund, Sweden.
authors.forename: J C S
authors.initials: JC
authors.lastname: Ho
authors.affiliation: Department of Microbiology, Immunology and Glycobiology (MIG), Institute of Laboratory Medicine, Lund University, Lund, Sweden.
authors.forename: A
authors.initials: A
authors.lastname: Nadeem
authors.affiliation: Department of Microbiology, Immunology and Glycobiology (MIG), Institute of Laboratory Medicine, Lund University, Lund, Sweden.
authors.forename: A
authors.initials: A
authors.lastname: Rydström
authors.affiliation: Department of Microbiology, Immunology and Glycobiology (MIG), Institute of Laboratory Medicine, Lund University, Lund, Sweden.
authors.forename: M
authors.initials: M
authors.lastname: Puthia
authors.affiliation: Department of Microbiology, Immunology and Glycobiology (MIG), Institute of Laboratory Medicine, Lund University, Lund, Sweden.
authors.forename: C
authors.initials: C
authors.lastname: Svanborg
mesh_terms: D000251:Adenosine Triphosphatases / Q000187:drug effects*; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D002352:Carrier Proteins / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D020558:GTP Phosphohydrolases / Q000187:drug effects*; D064593:Heterografts; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D007768:Lactalbumin / Q000494:pharmacology*; D051379:Mice; D018613:Microscopy, Confocal; D008958:Models, Molecular; D009711:Nucleotides / Q000378:metabolism; D009829:Oleic Acids / Q000494:pharmacology*; D040081:Protein Array Analysis; D011494:Protein Kinases / Q000187:drug effects*; D014407:Tumor Cells, Cultured
chemical_list: D000970:Antineoplastic Agents; D002352:Carrier Proteins; C506422:HAMLET complex, human; D009711:Nucleotides; D009829:Oleic Acids; D007768:Lactalbumin; D011494:Protein Kinases; D000251:Adenosine Triphosphatases; D020558:GTP Phosphohydrolases