Specificities of autoinhibitory domain peptides for four protein kinases: Implications for intact cell studies of protein kinase function

M. Kevin Smith, Roger J. Colbran, Thomas R. Soderling

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141 Scopus citations


Synthetic peptides corresponding to the autoinhibitory domains of calcium/calmodulin-dependent protein kinase II (CaMK-(281-309)), smooth muscle myosin light chain kinase (MLCK-(480-501)), and protein kinase C (PKC-(19-36)) as well as a peptide derived from the heat-stable inhibitor of cAMP-dependent protein kinase (PKI-tide) were tested for their inhibitory specificities. The inhibitory potencies of the four peptides were determined for each of the four protein kinases using both peptide substrates (at approximate Km, concentrations) and protein substrates (at concentrations less than Km). In agreement with previous studies PKI-tide was a specific and potent inhibitor of only cAMP kinase, and none of the other inhibitory peptides gave significant inhibition of cAMP kinase at concentrations of less than 100 μM. With synthetic peptide substrates, PKC-(19-36) strongly inhibited native PKC (IC50 < 1 μM) but also significantly inhibited autophosphorylated CaMK-II (IC50 = 30 μM) and proteolytically activated MLCK (IC50 = 35 μM). MLCK-(480-501) potently inhibited MLCK (IC50 = 0.25 μM) and also strongly inhibited both PKC and CaMK-II (IC50 = 1.4 and 1.7 μM, respectively). CaMK-(281-309) inhibited autophosphorylated CaMK-II, PKC, and proteolyzed MLCK almost equally (IC50 = 10, 38, and 48 μM, respectively). Qualitatively similar results were obtained with protein substrates. These studies validate the use of PKI-tide as a specific inhibitor of cAMP kinase in intact cell studies and suggest that PKC-(19-36) can also be used but only within a narrow concentration range. However, the autoinhibitory domain peptides from MLCK and CaMK-II are not sufficiently specific to be used in similar investigations.

Original languageEnglish (US)
Pages (from-to)1837-1840
Number of pages4
JournalJournal of Biological Chemistry
Issue number4
StatePublished - Feb 5 1990
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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