Preservation of the pattern of tyrosine phosphorylation in human neutrophil lysates: II. A sequential lysis protocol for the analysis of tyrosine phosphorylation-dependent signalling

• Published in: Journal of immunological methods Vol. 261; no. 1; pp. 85 - 101
• Main Authors: Gilbert, Caroline, Rollet-Labelle, Emmanuelle, Naccache, Paul H.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 01.03.2002; Elsevier
• Subjects: Adult; Biological and medical sciences; Buffers; Cbl; CD32; Cell Fractionation; Coenzymes - metabolism; Detergent insolubility; Diverse techniques; Fcγ receptors; fMet-Leu-Phe; Fundamental and applied biological sciences. Psychology; Fundamental immunology; GM-CSF; Granulocyte-Macrophage Colony-Stimulating Factor - pharmacology; Humans; Immunobiology; In Vitro Techniques; Kinase activity; Lyn; Microbiology; Molecular and cellular biology; monosodium urate; Monosodium urate crystals; Myeloid cells: ontogeny, maturation, markers, receptors; N-Formylmethionine Leucyl-Phenylalanine - pharmacology; Neutrophil; Neutrophils - drug effects; Neutrophils - metabolism; Osmolar Concentration; Phagocytosis; Phosphoproteins - isolation & purification; Phosphoproteins - metabolism; Phosphorylation; Polynuclears; Protein-Tyrosine Kinases - metabolism; Signal Transduction; Solubility; src-Family Kinases - metabolism; Syk; tyrosine; Tyrosine - metabolism; Tyrosine phosphorylation; Uric Acid - pharmacology; Kinase activity; Lyn; Fcγ receptors; Syk; Detergent insolubility; Cbl; MSU; RIPA; SB; Signal transduction; Monosodium urate crystals; GM-CSF; Tyrosine phosphorylation; HyperLB; LB; Neutrophil; fMet-Leu-Phe; Phagocytosis; ILB; CD32; HLB; Human; Tyrosine; Phosphorylation; Preservation; Stimulation; Signalling; Solubilization; Protein; Relevance; Enzymatic activity; Aminoacid; Lysis; Concentrate; Cell
• ISSN: 0022-1759; 1872-7905
• DOI: 10.1016/S0022-1759(01)00553-1

In stimulated neutrophils, the majority of tyrosine phosphorylated proteins are concentrated in Triton X-100 or NP-40 insoluble fractions. Most immunobiochemical studies, whose objective is to study the functional relevance of tyrosine phosphorylation are, however, performed using the supernatants of cells lysed in non-ionic detergent-containing buffers (RIPA lysis buffers). This observation prompted us to develop an alternative lysis protocol. We established a procedure involving the sequential lysis of neutrophils in buffers of increasing tonicities that not only preserved and solubilized tyrosine phosphorylated proteins but also retained their enzymatic activities. The sequential lysis of neutrophils in hypotonic, isotonic and hypertonic buffers containing non-ionic detergents resulted in the solubilisation of a significant fraction of tyrosine phosphorylated proteins. Furthermore, we observed that in monosodium urate crystals-stimulated neutrophils, Lyn activity was enhanced in the soluble fraction recovered from the hypertonic fraction, but not from that of the first hypotonic lysis. The distribution of tyrosine phosphorylated proteins between the NP-40 soluble and insoluble fractions was both substrate- and agonist-dependent. In neutrophils stimulated with fMet-Leu-Phe, MSU crystals or by CD32 ligation, the tyrosine phosphorylated proteins were mostly insoluble. On the other hand, in GM-CSF-treated cells, the phosphoproteins were more equally distributed between the two fractions. The results of this study provide a new experimental procedure for the investigation of tyrosine phosphorylation pathways in activated human neutrophils which may also be applicable to other cell types.