Characterization of growth factor-induced serine phosphorylation of tumor necrosis factor-alpha converting enzyme and of an alternatively translated polypeptide.
Tumor necrosis factor-alpha converting enzyme (TACE) is a prototype member of the adamalysin family of transmembrane metalloproteases that effects ectodomain cleavage and release of many transmembrane proteins, including transforming growth factor-alpha. Growth factors that act through tyrosine kinase receptors, as well as other stimuli, induce shedding through activation of the Erk mitogen-activated protein (MAP) kinase pathway without the need of new protein synthesis. How MAP kinase regulates shedding by TACE is not known. We now report that the cytoplasmic domain of TACE is phosphorylated in response to growth factor stimulation. We also identified a naturally expressed smaller polypeptide corresponding to most of the cytoplasmic domain of TACE. This protein, which we named SPRACT, is derived through alternative translation of the TACE-coding sequence and is, similarly to TACE, phosphorylated in response to growth factor and phorbol 12-myristate 13-acetate stimulation. Phosphoamino acid analysis revealed that growth factor-induced phosphorylation of TACE occurs only on serine and not on threonine or tyrosine. Tryptic mapping experiments coupled with site-directed mutagenesis identified Ser(819) as the major target of growth factor-induced phosphorylation, whereas Ser(791) undergoes dephosphorylation in response to growth factor stimulation. The phosphorylation of Ser(819), but not the dephosphorylation of Ser(791), depends on activation of the Erk MAP kinase pathway. Increased SPRACT expression or mutation of the TACE cytoplasmic domain to inactivate growth factor-induced phosphorylation did not detectably affect growth factor-induced shedding of transmembrane transforming growth factor-alpha by TACE. The roles of SPRACT and the cytoplasmic phosphorylation of TACE remain to be defined.
Fan
Huizhou
H
Department of Growth and Development, and Anatomy, Programs in Cell Biology and Developmental Biology, University of California, San Francisco, California 94143, USA. fanhu@umdnj.edu
Turck
Christoph W
CW
Derynck
Rik
R
D051722:ADAM Proteins; D000072198:ADAM17 Protein; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D003593:Cytoplasm / Q000378:metabolism; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D020935:MAP Kinase Signaling System; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D019606:Phosphoamino Acids / Q000737:chemistry; D010766:Phosphorylation; D011233:Precipitin Tests; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary; D012694:Serine / Q000737:chemistry / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection; D016211:Transforming Growth Factor alpha / Q000378:metabolism; D014357:Trypsin / Q000494:pharmacology
D010455:Peptides; D019606:Phosphoamino Acids; D016211:Transforming Growth Factor alpha; D012694:Serine; D014357:Trypsin; D051722:ADAM Proteins; D008666:Metalloendopeptidases; D000072198:ADAM17 Protein; C000606462:ADAM17 protein, human; D013755:Tetradecanoylphorbol Acetate