AKT1 and PTEN show the highest affinities among phosphoinositide binding proteins for the second messengers PtdIns(3,4,5)P3 and PtdIns(3,4)P2

Nelson, Nina; Razeto, Adelia; Gilardi, Alessia; Grättinger, Mira; Kirchmair, Johannes; Jücker, Manfred

doi:10.1016/j.bbrc.2021.06.027

Cited by 5 publications

(11 citation statements)

References 30 publications

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“…Despite reports of PIP lipid interactions being mediated by both the pleckstrin-homology related (PH-R) (13) or C2 domains (14), our in vitro single molecule dwell time analysis indicates that SHIP1 does not strongly associate with PI(4,5)P 2 , PI(3,4)P 2 , PI(3,4,5)P 3 , or PS lipids that are incorporated into supported membranes (Figure 1F). Based on the reported affinity that SHIP1 has for PI(3,4)P 2 (K D = 6 nM) and PI(3,4,5)P 3 (K D = 1 nM) (15), we expected to times (t 1 = 9 ms, t 2 = 56 ms, a= 44%, Figure 3G, Table S1). In the presence of lower ionic strength buffer, we also observed a modest increase in bulk membrane localization of mNG-SHIP1(PH-PP-C2) that was dependent on PI(3,4,5)P 3 and PS lipids (Figure 3H).…”

Section: Discussionmentioning

confidence: 99%

“…Based on reported lipid interactions, researchers have hypothesized that SHIP1 catalyzes the dephosphorylation of PI(3,4,5)P 3 with a positive feedback loop based on interactions with both substrate and product (13)(14)(15). To measure potentially nonlinear reaction kinetics, we developed an in vitro assay to simultaneously visualize SHIP1 membrane binding and lipid phosphatase activity using TIRF microscopy.…”

Section: Ship1 Catalyzes Dephosphorylation Of Pi(345)p 3 With Unimole...mentioning

confidence: 99%

“…SHIP1 contains two proposed lipid binding domains that flank the central phosphatase domain and have been proposed to regulate membrane localization and catalytic activity (Figure 1A). The pleckstrinhomology related (PH-R) domain was shown to interact with the substrate of SHIP1, PI(3,4,5)P 3 , while the C2 domain reportedly binds to the product of SHIP1, PI(3,4)P 2 (13)(14)(15). Mutations in the PH-R domain disrupted localization to the phagocytic cup during FcγR-mediated phagocytosis in macrophages but did not affect SHIP1 catalysis.…”

Section: Introductionmentioning

confidence: 99%

“…To date, no biochemical studies have directly visualized the interactions between purified SHIP1 and proposed PIP lipid binding partners. Previous experiments have relied on surface plasma resonance (SPR) (15) and protein lipid overlay (PLO) assays (Ong et al 2007, Ming-Lum et al 2012) to measure SHIP1 lipid interactions. Given the combination of protein and lipid interactions that potentially regulate SHIP1, it remains unclear whether SHIP1's membrane localization is controlled directly or indirectly by PIP lipid interactions.…”

Section: Introductionmentioning

confidence: 99%

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Mechanisms controlling membrane recruitment and activation of autoinhibited SHIP1

Waddell

Drew

Rupp

et al. 2023

Preprint

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Signal transduction downstream of growth factor and immune receptor activation relies on the production of phosphatidylinositol-(3,4,5)-trisphosphate (PI(3,4,5)P3) lipids by phosphoinositide-3-kinase (PI3K). Regulating the strength and duration of PI3K signaling in immune cells, Src homology 2 domain-containing inositol 5-phosphatase 1 (SHIP1) controls the dephosphorylation of PI(3,4,5)P3 to generate PI(3,4)P2. Although SHIP1 has been shown to regulate neutrophil chemotaxis, B-cell signaling, and cortical oscillations in mast cells, the role that lipid and protein interactions serve in controlling SHIP1 membrane recruitment and activity remains unclear. Using single molecule TIRF microscopy, we directly visualized membrane recruitment and activation of SHIP1 on supported lipid bilayers and the cellular plasma membrane. We find that SHIP1's interactions with lipids are insensitive to dynamic changes in PI(3,4,5)P3 both in vitro and in vivo. Very transient SHIP1 membrane interactions were detected only when membranes contained a combination of phosphatidylserine (PS) and PI(3,4,5)P3 lipids. Molecular dissection reveals that SHIP1 is autoinhibited with the N-terminal SH2 domain playing a critical role in suppressing phosphatase activity. Robust SHIP1 membrane localization and relief of autoinhibition can be achieved through interactions with immunoreceptor derived phosphopeptides presented either in solution or conjugated to supported membranes. Overall, this work provides new mechanistic details concerning the dynamic interplay between lipid binding specificity, protein-protein interactions, and activation of autoinhibited SHIP1.

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Section: Discussionmentioning

confidence: 99%

Section: Ship1 Catalyzes Dephosphorylation Of Pi(345)p 3 With Unimole...mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Mechanisms controlling membrane recruitment and activation of autoinhibited SHIP1

Waddell

Drew

Rupp

et al. 2023

Preprint

View full text Add to dashboard Cite

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“…However, due to low transfection efficiency, the effect of gene therapy has not been fully achieved. The key to improving the effect of gene therapy lies in the carrier system [ 12 ]. Therefore, the development of an efficient, specific and safe carrier system that is able to directly transfer biologically effective proteins into tumor cells (especially living tissue cells) to inhibit or kill tumors has become a research hotspot in China and other countries.…”

Section: Discussionmentioning

confidence: 99%

Effect of CTP-mediated PTEN on 5637 bladder cancer cells and the underlying molecular mechanism

Yuan

Yang

et al. 2022

BMC Urol

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Objective The aim of the present study was to explore the effect of cytoplasmic transduction peptide (CTP)-phosphatase and tensin homolog (PTEN) on the proliferation, cell cycle, apoptosis, migration and invasion of bladder cancer cells and the underlying molecular mechanism. Methods A eukaryotic expression vector, pTT5-CTP-PTEN, was constructed. The constructed vector was transfected into HEK 293-6E cells to express a fusion protein, CTP-PTEN. The fusion protein was purified. 5637 bladder cancer cells were cocultured with purified CTP-PTEN fusion protein. Target gene expression, protein expression, cell proliferation, cell cycle, apoptosis, cell invasion and cell migration were examined by reverse transcription polymerase chain reaction (RT-PCR), western blot, MTT assay, flow cytometry, Transwell assay, and cell scratch assay, respectively. Results Both PTEN and CTP-PTEN fusion protein inhibited the proliferation, cell cycle, invasion and migration of bladder cancer cells and promoted the apoptosis of bladder cancer cells. The effect of CTP-PTEN was more significant. Conclusions The fused expression of CTP and PTEN significantly increased the penetrability of the tumor suppressor gene PTEN into cancer cells. The CTP-PTEN fusion protein exhibited a significant carcinostatic effect on 5637 bladder cancer cells.

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Mechanisms controlling membrane recruitment and activation of the autoinhibited SHIP1 inositol 5-phosphatase

Waddell

Drew

Rupp

et al. 2023

Journal of Biological Chemistry

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AKT1 and PTEN show the highest affinities among phosphoinositide binding proteins for the second messengers PtdIns(3,4,5)P3 and PtdIns(3,4)P2

Cited by 5 publications

References 30 publications

Mechanisms controlling membrane recruitment and activation of autoinhibited SHIP1

Mechanisms controlling membrane recruitment and activation of autoinhibited SHIP1

Effect of CTP-mediated PTEN on 5637 bladder cancer cells and the underlying molecular mechanism

Mechanisms controlling membrane recruitment and activation of the autoinhibited SHIP1 inositol 5-phosphatase

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