Rap1A is a substrate for cyclic AMP-dependent protein kinase in human neutrophils.
The Ras-related protein, Rap1B, has previously been shown to serve as a PKA substrate in vitro and to be phosphorylated by cAMP elevating agents in human platelets. We have purified a Rap1 protein that serves as a PKA substrate from human neutrophils, and we now identify this protein as Rap1A. A 23-kDa protein that co-migrated with recombinant Rap1A was phosphorylated in electroporated human neutrophils upon stimulation by cAMP in the presence of [gamma-32P]ATP. This protein could be immunoprecipitated by the Rap1A/B-specific antibody, R61. The 23-kDa phosphoprotein was monitored during the purification of Rap1 from neutrophil membrane extracts and was shown to copurify with Rap1 during the DEAE Sephacel, heptylamine Sepharose, and MonoQ chromatography steps utilized. The purified protein was phosphorylated to an extent of 1 mol phosphate/mol GTP gamma S bound. This protein was identified as Rap1A by: 1) amino acid sequence analysis; and 2) immunoblotting with a Rap1A-specific antibody. The amino acid phosphorylated on Rap1A by PKA was a serine residue. The site of phosphorylation was indicated by carboxypeptidase digestion and confirmed using a mutant recombinant Rap1A lacking the relevant serine (serine-180). Rap1A, not Rap1B, appears to be the major 23-kDa PKA substrate in human neutrophils. It is possible that Rap1A plays a role in human neutrophils in mediating the inhibitory effects of cAMP-elevating agents upon chemoattractant-stimulated cell activation.
Quilliam
L A
LA
Research Institute of Scripps Clinic, La Jolla, CA.
Mueller
H
H
Bohl
B P
BP
Prossnitz
V
V
Sklar
L A
LA
Der
C J
CJ
Bokoch
G M
GM
D015180:Electrophoresis, Gel, Two-Dimensional; D019204:GTP-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D009504:Neutrophils / Q000737:chemistry / Q000201:enzymology*; D010455:Peptides; D011494:Protein Kinases / Q000502:physiology*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D014862:Wasp Venoms / Q000494:pharmacology; D020668:rap GTP-Binding Proteins
D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; D011518:Proto-Oncogene Proteins; D014862:Wasp Venoms; D012694:Serine; C037500:mastoparan; D011494:Protein Kinases; D019204:GTP-Binding Proteins; D020668:rap GTP-Binding Proteins