Rap1A is a substrate for cyclic AMP-dependent protein kinase in human neutrophils

L. A. Quilliam, H. Mueller, B. P. Bohl, V. Prossnitz, L. A. Sklar, C. J. Der, G. M. Bokoch

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Abstract

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 [γ-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 GTPγS 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.

Original languageEnglish (US)
Pages (from-to)1628-1635
Number of pages8
JournalJournal of Immunology
Volume147
Issue number5
StatePublished - Jan 1 1991

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Cyclic AMP-Dependent Protein Kinases
Neutrophils
Serine
Proteins
rap1 GTP-Binding Proteins
Carboxypeptidases
ras Proteins
Agarose Chromatography
Antibodies
Phosphoproteins
Chemotactic Factors
Protein Sequence Analysis
Immunoblotting
Digestion
Blood Platelets
Adenosine Triphosphate
Phosphates
Phosphorylation
Amino Acids
Membranes

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology

Cite this

Quilliam, L. A., Mueller, H., Bohl, B. P., Prossnitz, V., Sklar, L. A., Der, C. J., & Bokoch, G. M. (1991). Rap1A is a substrate for cyclic AMP-dependent protein kinase in human neutrophils. Journal of Immunology, 147(5), 1628-1635.

Rap1A is a substrate for cyclic AMP-dependent protein kinase in human neutrophils. / Quilliam, L. A.; Mueller, H.; Bohl, B. P.; Prossnitz, V.; Sklar, L. A.; Der, C. J.; Bokoch, G. M.

In: Journal of Immunology, Vol. 147, No. 5, 01.01.1991, p. 1628-1635.

Research output: Contribution to journalArticle

Quilliam, LA, Mueller, H, Bohl, BP, Prossnitz, V, Sklar, LA, Der, CJ & Bokoch, GM 1991, 'Rap1A is a substrate for cyclic AMP-dependent protein kinase in human neutrophils', Journal of Immunology, vol. 147, no. 5, pp. 1628-1635.
Quilliam LA, Mueller H, Bohl BP, Prossnitz V, Sklar LA, Der CJ et al. Rap1A is a substrate for cyclic AMP-dependent protein kinase in human neutrophils. Journal of Immunology. 1991 Jan 1;147(5):1628-1635.
Quilliam, L. A. ; Mueller, H. ; Bohl, B. P. ; Prossnitz, V. ; Sklar, L. A. ; Der, C. J. ; Bokoch, G. M. / Rap1A is a substrate for cyclic AMP-dependent protein kinase in human neutrophils. In: Journal of Immunology. 1991 ; Vol. 147, No. 5. pp. 1628-1635.
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