PubMed Journals: Biochim Biophys Acta

  Source:		PMID: 1998697

    		Biochim Biophys Acta. 1991 Jan 30;1061(2):253-66.
			Phosphorylation sites in human erythrocyte
			band 3 protein.

			Yannoukakos D(1), Vasseur C, Piau JP, Wajcman
			H, Bursaux E.

			Author Information
			(1) INSERM U299, Hôpital de Bicêtre, Le
			Kremlin-Bicêtre, France.

			The human red cell anion-exchanger, band
			3 protein, is one of the main phosphorylated
			proteins of the erythrocyte membrane. Previous
			studies from this laboratory have shown
			that ATP-depletion of the red blood cell
			decreased the anion-exchange rate, suggesting
			that band 3 protein phosphorylation could
			be involved in the regulation of anion transport
			function (Bursaux et al. (1984) Biochim.
			Biophys. Acta 777, 253-260). Phosphorylation
			occurs mainly on the cytoplasmic domain
			of the protein and the major site of phosphorylation
			was assigned to tyrosine-8 (Dekowski et
			al. (1983) J. Biol. Chem. 258, 2750-2753).
			This site being very far from the integral,
			anion-exchanger domain, the aim of the present
			study was to determine whether phosphorylation
			sites exist in the integral domain. The
			phosphorylation reaction was carried out
			on isolated membranes in the presence of
			[gamma-32P]ATP and phosphorylated band 3
			protein was then isolated. Both the cytoplasmic
			and the membrane spanning domains were purified.
			The predominant phosphorylation sites were
			found on the cytoplasmic domain. RP-HPLC
			analyses of the tryptic peptides of whole
			band 3 protein, and of the isolated cytoplasmic
			and membrane-spanning domains allowed for
			the precise localization of the phosphorylated
			residues. 80% of the label was found in
			the N-terminal tryptic peptide (T-1), (residues
			1-56). In this region, all the residues
			susceptible to phosphorylation were labeled
			but in varying proportion. Under our conditions,
			the most active membrane kinase was a tyrosine
			kinase, activated preferentially by Mn2+
			but also by Mg2+. Tyrosine-8 was the main
			phosphate acceptor residue (50-70%) of the
			protein, tyrosine-21 and tyrosine-46 residues
			were also phosphorylated but to a much lesser
			extent. The main targets of membrane casein
			kinase, preferentially activated by Mg2+,
			were serine-29, serine-50, and threonine(s)-39,
			-42, -44, -48, -49, -54 residue(s) located
			in the T-1 peptide. A tyrosine phosphatase
			activity was copurified with whole band
			3 protein which dephosphorylates specifically
			P-Tyr-8, indicating a highly exchangeable
			phosphate. The membrane-spanning fragment
			was only faintly labeled.

			PMID: 1998697 [Indexed for MEDLINE]

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