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Genome-wide screen of gamma-secretase-mediated intramembrane cleavage of receptor tyrosine kinases.

ABSTRACT: Receptor tyrosine kinases (RTKs) have been demonstrated to signal via regulated intramembrane proteolysis, in which ectodomain shedding and subsequent intramembrane cleavage by gamma-secretase leads to release of a soluble intracellular receptor fragment with functional activity. For most RTKs, however, it is unknown whether they can exploit this new signaling mechanism. Here we used a system-wide screen to address the frequency of susceptibility to gamma-secretase cleavage among human RTKs. The screen covering 45 of the 55 human RTKs identified 12 new as well as all nine previously published gamma-secretase substrates. We biochemically validated the screen by demonstrating that the release of a soluble intracellular fragment from endogenous AXL was dependent on the sheddase disintegrin and metalloprotease 10 (ADAM10) and the gamma-secretase component presenilin-1. Functional analysis of the cleavable RTKs indicated that proliferation promoted by overexpression of the TAM family members AXL or TYRO3 depends on gamma-secretase cleavage. Taken together, these data indicate that gamma-secretase-mediated cleavage provides an additional signaling mechanism for numerous human RTKs.

SUBMITTER: Merilahti JAM

PROVIDER: S-EPMC5662267 | biostudies-other | 2017 Nov

REPOSITORIES: biostudies-other

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Genome-wide screen of gamma-secretase-mediated intramembrane cleavage of receptor tyrosine kinases.

Merilahti Johannes A M JAM Ojala Veera K VK Knittle Anna M AM Pulliainen Arto T AT Elenius Klaus K

Molecular biology of the cell 20170913 22

Receptor tyrosine kinases (RTKs) have been demonstrated to signal via regulated intramembrane proteolysis, in which ectodomain shedding and subsequent intramembrane cleavage by gamma-secretase leads to release of a soluble intracellular receptor fragment with functional activity. For most RTKs, however, it is unknown whether they can exploit this new signaling mechanism. Here we used a system-wide screen to address the frequency of susceptibility to gamma-secretase cleavage among human RTKs. The ...[more]

PMID: 28904208

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Project description:BackgroundAlcadein proteins (Alcs; Alc?, Alc?and Alc?) are predominantly expressed in neurons, as is Alzheimer's ?-amyloid (A?) precursor protein (APP). Both Alcs and APP are cleaved by primary ?- or ?-secretase to generate membrane-associated C-terminal fragments (CTFs). Alc CTFs are further cleaved by ?-secretase to secrete p3-Alc peptide along with the release of intracellular domain fragment (Alc ICD) from the membrane. In the case of APP, APP CTF? is initially cleaved at the ?-site to release the intracellular domain fragment (AICD) and consequently the ?-site is determined, by which A? generates. The initial ?-site is thought to define the final ?-site position, which determines whether A?40/43 or A?42 is generated. However, initial intracellular ?-cleavage sites of Alc CTF to generate Alc ICD and the molecular mechanism that final ?-site position is determined remains unclear in Alcs.MethodologyUsing HEK293 cells expressing Alcs plus presenilin 1 (PS1, a catalytic unit of ?-secretase) and the membrane fractions of these cells, the generation of p3-Alc possessing C-terminal ?-cleavage site and Alc ICD possessing N-terminal ?-cleavage site were analysed with MALDI-TOF/MS. We determined the initial ?-site position of all Alc?, Alc? and Alc?, and analyzed the relationship between the initially determined ?-site position and the final ?-cleavage position.ConclusionsThe initial ?-site position does not always determine the final ?-cleavage position in Alcs, which differed from APP. No additional ?-cleavage sites are generated from artificial/non-physiological positions of ?-cleavage for Alcs, while the artificial ?-cleavage positions can influence in selection of physiological ?-site positions. Because alteration of ?-secretase activity is thought to be a pathogenesis of sporadic Alzheimer's disease, Alcs are useful and sensitive substrate to detect the altered cleavage of substrates by ?-secretase, which may be induced by malfunction of ?-secretase itself or changes of membrane environment for enzymatic reaction.

Project description:BACKGROUND:γ-Secretase is a multiprotein protease that cleaves amyloid protein precursor (APP) and other type I transmembrane proteins. It has two catalytic subunits, presenilins 1 and 2 (PS1 and 2). In our previous report, we observed subtle differences in PS1- and PS2-mediated cleavages of select substrates and slightly different potencies of PS1 versus PS2 inhibition for select γ-secretase inhibitors (GSIs) on various substrates. In this study, we investigated whether γ-secretase modulators (GSMs) and inverse γ-secretase modulators (iGSMs) modulate γ-secretase processivity using multiple different substrates. We next used HEK 293T cell lines in which PSEN1 or PSEN2 was selectively knocked out to investigate processivity and response to GSMs and iGSMs. METHODS:For cell-free γ-secretase cleavage assay, recombinant substrates were incubated with CHAPSO-solubilized CHO or HEK 293T cell membrane with GSMs or iGSMs in suitable buffer. For cell-based assay, cDNA encoding substrates were transfected into HEK 293T cells. Cells were then treated with GSMs or iGSMs, and conditioned media were collected. Aβ and Aβ-like peptide production from cell-free and cell-based assay were measured by ELISA and mass spectrometry. RESULT:These studies demonstrated that GSMs are highly selective for effects on APP, whereas iGSMs have a more promiscuous effect on many substrates. Surprisingly, iGSMs actually appear to act as like GSIs on select substrates. The data with PSEN1 or PSEN2 knocked out HEK 293T reveal that PS1 has higher processivity and response to GSMs than PS2, but PS2 has higher response to iGSM. CONCLUSION:Collectively, these data indicate that GSMs are likely to have limited target-based toxicity. In addition, they show that iGSMs may act as substrate-selective GSIs providing a potential new route to identify leads for substrate-selective inhibitors of certain γ-secretase-mediated signaling events. With growing concerns that long-term β-secretase inhibitor is limited by target-based toxicities, such data supports continued development of GSMs as AD prophylactics.

Dataset Information

Genome-wide screen of gamma-secretase-mediated intramembrane cleavage of receptor tyrosine kinases.

Publications

Genome-wide screen of gamma-secretase-mediated intramembrane cleavage of receptor tyrosine kinases.

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