title: TPCK inhibits AGC kinases by direct activation loop adduction at phenylalanine-directed cysteine residues.
abstract: N-alpha-tosyl-L-phenylalanyl chloromethyl ketone (TPCK) has anti-tumorigenic properties, but its direct cellular targets are unknown. Previously, we showed TPCK inhibited the PDKl-dependent AGC kinases RSK, Akt and S6K1 without inhibiting PKA, ERK1/2, PI3K, and PDK1 itself. Here we show TPCK-inhibition of the RSK-related kinases MSK1 and 2, which can be activated independently of PDK1. Mass spectrometry analysis of RSK1, Aktl, S6K1 and MSK1 immunopurified from TPCK-treated cells identified TPCK adducts on cysteines located in conserved activation loop Phenylalanine-Cysteine (Phe-Cys) motifs. Mutational analysis of the Phe-Cys residues conferred partial TPCK resistance. These studies elucidate a primary mechanism by which TPCK inhibits several AGC kinases, inviting consideration of TPCK-like compounds in chemotherapy given their potential for broad control of cellular growth, proliferation and survival.
authors.affiliation: Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Rana
authors.initials: R
authors.lastname: Anjum
authors.forename: Eunice
authors.initials: E
authors.lastname: Pae
authors.forename: John
authors.initials: J
authors.lastname: Blenis
authors.forename: Bryan A
authors.initials: BA
authors.lastname: Ballif
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001483:Base Sequence; D017124:Conserved Sequence; D003545:Cysteine / Q000737:chemistry; D017931:DNA Primers / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000737:chemistry; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D011494:Protein Kinases / Q000737:chemistry* / Q000235:genetics; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D038744:Ribosomal Protein S6 Kinases, 90-kDa / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D014108:Tosylphenylalanyl Chloromethyl Ketone / Q000494:pharmacology*
chemical_list: D017931:DNA Primers; C000631429:PDK1 protein, human; D047428:Protein Kinase Inhibitors; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D014108:Tosylphenylalanyl Chloromethyl Ketone; D010649:Phenylalanine; D011494:Protein Kinases; C494918:AKT1 protein, human; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; C069329:RPS6KA1 protein, human; C115634:RPS6KA4 protein, human; D038744:Ribosomal Protein S6 Kinases, 90-kDa; C116919:mitogen and stress-activated protein kinase 1; D003545:Cysteine