Characterization of TAT-mediated transport of detachable kinase substrates.
The conjugation of peptides derived from the HIV TAT protein to membrane-impermeant molecules has gained wide acceptance as a means for intracellular delivery. Numerous studies have addressed the mechanism of uptake and kinetics of TAT translocation, but the cytosolic concentrations and bioavailability of the transported cargo have not been well-characterized. The current paper utilizes a microanalytical assay to perform quantitative single-cell measurements of the concentration and accessibility of peptide-based substrates for protein kinase B (PKB) and Ca(2+)/calmodulin-activated kinase II. The substrate peptide and TAT were conjugated through a releasable linker, either a disulfide or photolabile bond. Free substrate peptide concentrations of approximately 10(-20)-10(-18) moles were attainable in a cell when substrates were delivered utilizing these conjugates. The substrate peptides delivered as a disulfide conjugate were often present in the cytosol as several oxidized forms. Brief exposure of cells loaded with the photolabile conjugates to UVA light released free substrate peptide into the cytosol. Substrate peptide delivered by either conjugate was accessible to cytosolic kinase as demonstrated by the efficient phosphorylation of the peptide when the appropriate kinase was active. After incubation of the conjugated substrate with cells, free, kinase-accessible substrate was detectable in less than 30 min. Release of the majority of loaded substrate peptide from sequestered organelles occurred within 1 h. The utility of the photocleavable conjugates was demonstrated by measuring the activation of PKB in 3T3 cells after addition of varying concentrations of platelet-derived growth factor.
Soughayer
Joseph S
JS
Department of Physiology and Biophysics, College of Medicine, University of California, Irvine, California 92697, USA.
Wang
Yan
Y
Li
Huaina
H
Cheung
Shing-Hu
SH
Rossi
Frank M
FM
Stanbridge
Eric J
EJ
Sims
Christopher E
CE
Allbritton
Nancy L
NL
D000818:Animals; D001692:Biological Transport; D015153:Blotting, Western; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002460:Cell Line; D045744:Cell Line, Tumor; D003600:Cytosol / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D019075:Electrophoresis, Capillary; D004789:Enzyme Activation; D019793:Fluorescein / Q000494:pharmacology; D015696:Gene Products, tat / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D041681:NIH 3T3 Cells; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D010770:Phosphotransferases / Q000737:chemistry*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D051381:Rats; D013696:Temperature; D013997:Time Factors; D014466:Ultraviolet Rays; D048051:p38 Mitogen-Activated Protein Kinases
D015696:Gene Products, tat; D010455:Peptides; D011518:Proto-Oncogene Proteins; D010770:Phosphotransferases; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D019793:Fluorescein