TPCK inhibits AGC kinases by direct activation loop adduction at phenylalanine-directed cysteine residues

FEBS Lett. 2012 Sep 21;586(19):3471-6. doi: 10.1016/j.febslet.2012.07.075. Epub 2012 Aug 10.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Motifs
  • Amino Acid Sequence
  • Amino Acid Substitution
  • Base Sequence
  • Conserved Sequence
  • Cysteine / chemistry
  • DNA Primers / genetics
  • HEK293 Cells
  • Humans
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Phenylalanine / chemistry
  • Protein Kinase Inhibitors / pharmacology*
  • Protein Kinases / chemistry*
  • Protein Kinases / genetics
  • Protein Serine-Threonine Kinases / metabolism
  • Proto-Oncogene Proteins c-akt / antagonists & inhibitors
  • Proto-Oncogene Proteins c-akt / chemistry
  • Proto-Oncogene Proteins c-akt / genetics
  • Pyruvate Dehydrogenase Acetyl-Transferring Kinase
  • Ribosomal Protein S6 Kinases, 90-kDa / antagonists & inhibitors
  • Ribosomal Protein S6 Kinases, 90-kDa / chemistry
  • Ribosomal Protein S6 Kinases, 90-kDa / genetics
  • Sequence Homology, Amino Acid
  • Tosylphenylalanyl Chloromethyl Ketone / pharmacology*

Substances

  • DNA Primers
  • PDK1 protein, human
  • Protein Kinase Inhibitors
  • Pyruvate Dehydrogenase Acetyl-Transferring Kinase
  • Tosylphenylalanyl Chloromethyl Ketone
  • Phenylalanine
  • Protein Kinases
  • AKT1 protein, human
  • Protein Serine-Threonine Kinases
  • Proto-Oncogene Proteins c-akt
  • RPS6KA1 protein, human
  • RPS6KA4 protein, human
  • Ribosomal Protein S6 Kinases, 90-kDa
  • mitogen and stress-activated protein kinase 1
  • Cysteine