Abstract
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.
Highlights
Various cell surface proteins with diverse functions undergo regulated proteolytic cleavage of their extracellular domains, a process that results in ectodomain shedding
MDC9, which shares sequence similarity with Tumor necrosis factor-␣ converting enzyme (TACE) in its cytoplasmic domain, is phosphorylated by protein kinase C␦ [51]. In one study this phosphorylation was shown to regulate the induction of heparin binding-epidermal growth factor-like growth factor (HB-EGF) shedding by PMA [51], whereas other findings indicate that PMA may activate HB-EGF shedding through the Erk mitogen-activated protein (MAP) kinase signaling pathway instead [33, 35]
They were not competed out in immunoprecipitations in the presence of excess peptide immunogen (Fig. 1C). These results indicate that both the glycosylated pro-TACE and mature TACE are phosphorylated and that EGF, fibroblast growth factor (FGF), serum, and PMA, which are known to activate ectodomain shedding, rapidly induce phosphorylation of the TACE cytoplasmic domain
Summary
Growth factors and activated tyrosine kinase receptors rapidly induce ectodomain shedding of TGF-␣ and other transmembrane proteins through induction of the Erk MAP kinase signaling pathway without the need for new protein synthesis [32]. PMA-induced ectodomain shedding of TGF-␣ or its family member heparin binding-epidermal growth factor-like growth factor (HB-EGF) and TNF-␣ is mediated through activation of the Erk signaling pathway [32, 33, 35]. MDC9, which shares sequence similarity with TACE in its cytoplasmic domain, is phosphorylated by protein kinase C␦ [51] In one study this phosphorylation was shown to regulate the induction of HB-EGF shedding by PMA [51], whereas other findings indicate that PMA may activate HB-EGF shedding through the Erk MAP kinase signaling pathway instead [33, 35]. The function of these phosphorylation sites and the role of SPRACT remain to be characterized
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