Cloning of a fibroblast growth factor receptor 1 splice variant from Xenopus embryos that lacks a protein kinase C site important for the regulation of receptor activity.
A cDNA clone, predicted to encode a variant form of the type 1 fibroblast growth factor receptor (FGFR1) containing a dipeptide Val-Thr (VT) deletion at amino acid positions 423 and 424 located within the juxtamembrane region, was isolated from a Xenopus embryo (stage 8 blastula) library. Sequence analysis of genomic DNA encoding a portion of the FGFR1 juxtamembrane region demonstrated that this variant form arises from use of an alternative 5' splice donor site. RNase protection analysis revealed that both VT- and VT+ forms of the FGFR1 were expressed throughout embryonic development, the VT+ being the major form. Amino acid position 424 is located within a consensus sequence for phosphorylation by a number of Ser/Thr kinases. We demonstrate that a VT+ peptide was specifically phosphorylated by protein kinase C (PKC) in vitro, but not by protein kinase A (PKA). A VT- peptide, on the other hand, was not a substrate for either enzyme. Phosphorylation levels of in vitro synthesized FGFR-VT+ protein by PKC were twice that of FGFR-VT- protein. In a functional assay, Xenopus oocytes expressing FGFR-VT- or FGFR-VT+ protein were equally able to mobilize intracellular Ca2+ in response to basic fibroblast growth factor (bFGF). However, pretreatment with phorbol 12-myristate 13-acetate significantly reduced this mobilization in oocytes expressing FGFR-VT+ while having little effect on oocytes expressing FGFR-VT-. These findings demonstrate that alternative splicing of Val423-Thr424 generates isoforms which differ in their ability to be regulated by phosphorylation and thus represents an important mechanism for regulating FGFR activity.
Gillespie
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Terry Fox Cancer Research Laboratories, Division of Basic Medical Sciences, Faculty of Medicine, Memorial University of Newfoundland, St. John's, Canada.
Chen
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Paterno
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D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001755:Blastocyst / Q000502:physiology; D002118:Calcium / Q000378:metabolism; D003001:Cloning, Molecular; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D017931:DNA Primers; D004625:Embryo, Nonmammalian / Q000378:metabolism*; D005260:Female; D005346:Fibroblast Growth Factors / Q000378:metabolism; D014644:Genetic Variation; D015698:Genomic Library; D008648:Mesoderm / Q000502:physiology; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011493:Protein Kinase C / Q000378:metabolism*; D020794:Receptor Protein-Tyrosine Kinases; D051496:Receptor, Fibroblast Growth Factor, Type 1; D017468:Receptors, Fibroblast Growth Factor / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D014982:Xenopus laevis / Q000196:embryology
D017931:DNA Primers; D010455:Peptides; D017468:Receptors, Fibroblast Growth Factor; D011994:Recombinant Proteins; D005346:Fibroblast Growth Factors; D020794:Receptor Protein-Tyrosine Kinases; D051496:Receptor, Fibroblast Growth Factor, Type 1; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D002118:Calcium