Protein phosphorylation in neutrophils monitored with phosphospecific antibodies.
Protein phosphorylation in neutrophils was monitored with two phosphospecific antibodies (pAbs) [termed pPKC(S1) Ab and pPKC(S2) Ab] that recognize products of protein kinase C (PKC) and other Arg/Lys-directed Ser/Thr protein kinases. The pPKC(S1) Ab bound preferentially to p-Ser/p-Thr residues with Arg or Lys in the -3 and -5 positions or the -2 and -3 positions, whereas the pPKC(S2) Ab bound preferentially to p-Ser with Arg or Lys in the -2 and +2 positions and with a hydrophobic residue at the +1 position. Phosphorylated pleckstrin, myristoylated alanine-rich C-kinase substrate (MARCKS), the 47-kDa subunit of the phagocyte oxidase (p47-phox) and numerous unidentified proteins that underwent phosphorylation during neutrophil stimulation were readily detected with these pAbs. Priming effects of tumor necrosis factor alpha (TNF-alpha) and the susceptibility of certain reactions in neutrophils to inhibitors of protein kinases could also be easily investigated with these reagents. Compared to the commonly used 32P-labeling/autoradiographic method, Western blotting with pAbs was found to be a faster, safer, more specific and in many cases more sensitive approach for monitoring protein phosphorylation events in neutrophils. These pAbs may facilitate the identification of several new phosphorylation reactions involved in neutrophil stimulation.
Department of Anesthesiology, Brigham and Women's Hospital, Thorn Building, Room 703, 75 Francis Street, Boston, MA 02115, USA.
Taisuke
T
Ohira
Qian
Q
Zhan
Qingyuan
Q
Ge
Thomas
T
VanDyke
John A
JA
Badwey
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology*; D001665:Binding Sites; D001798:Blood Proteins / Q000378:metabolism*; D006168:Guinea Pigs; D006801:Humans; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000276:immunology / Q000502:physiology; D047428:Protein Kinase Inhibitors; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D000906:Antibodies; D001798:Blood Proteins; D047428:Protein Kinase Inhibitors; D014409:Tumor Necrosis Factor-alpha; D009240:N-Formylmethionine Leucyl-Phenylalanine; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate