Phage display based cloning of proteins interacting with the cytoplasmic tail of membrane immunoglobulins.
The reduced quantity and quality of serum immunoglobulins (sIgs) in mutant mice expressing truncated cytoplasmic tails of IgE and IgG1 indicate an active role for the cytoplasmic domains of mIgG1 and mIgE. We used phage display technology to identify candidate proteins able to interact with the cytoplasmic tail of mIgE. Using a murine cDNA B cell library displayed on the surface of phage as prey and the 28 amino acid long cytoplasmic tail of IgE as bait, we isolated phage encoding the murine hematopoietic progenitor kinase 1 (HPK1). Surface plasmon resonance analysis measurements confirmed affinity of HPK1 to the mIgE cytoplasmic tail and revealed association to other immunoglobulin isotypes as well. Immunoprecipitation experiments, using lysates from two B cell lines expressing nitrophenyl (NP) specific mIgE molecules showed co-precipitation of IgE and HPK1. The interaction of HPK1 with the cytoplasmic domains of membrane immunoglobulins indicate an active role of the tails as part of an isotype specific signal transduction, independent from the Igalpha/Igbeta heterodimers, and may represent a missing link to upstream regulatory elements of HPK1 activation.
Geisberger
Roland
R
Department of Genetics and General Biology, Hellbrunnerstrasse 34, A-5020 Salzburg, Austria.
Prlic
Martin
M
Achatz-Straussberger
Gertrude
G
Oberndorfer
Iris
I
Luger
Elke
E
Lamers
Marinus
M
Crameri
Reto
R
Appenzeller
Ulrich
U
Wienands
Jürgen
J
Breitenbach
Michael
M
Ferreira
Fatima
F
Achatz
Gernot
G
D000595:Amino Acid Sequence; D000818:Animals; D001402:B-Lymphocytes / Q000276:immunology; D003001:Cloning, Molecular; D018076:DNA, Complementary; D007073:Immunoglobulin E / Q000737:chemistry / Q000378:metabolism; D007132:Immunoglobulin Isotypes / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D008213:Lymphocyte Activation; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D019151:Peptide Library; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D010954:Plasmacytoma; D011233:Precipitin Tests; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011947:Receptors, Antigen, B-Cell / Q000737:chemistry / Q000378:metabolism*; D017455:Receptors, IgE / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020349:Surface Plasmon Resonance; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D007132:Immunoglobulin Isotypes; D019151:Peptide Library; D010455:Peptides; D011506:Proteins; D011947:Receptors, Antigen, B-Cell; D017455:Receptors, IgE; D007073:Immunoglobulin E; C102102:hematopoietic progenitor kinase 1; D017346:Protein Serine-Threonine Kinases