Phospho-Sin1 (Thr86) Antibody [L3G17]

Application: Reactivity: Human, Mouse

Usage Information

Dilution
WB1:1000 IP1:50

Application
WB, IP

Reactivity
Human, Mouse

Source
Rabbit Monoclonal Antibody

Storage Buffer
PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN3

Storage (from the date of receipt)
-20°C (avoid freeze-thaw cycles), 2 years

Predicted MW Observed MW
59 kDa 78 kDa, 74 kDa
^*Why do the predicted and actual molecular weights differ? The following reasons may explain differences between the predicted and actual protein molecular weight. Post-translational modifications(e.g., phosphorylation, glycosylation); Splice variants and isoforms; Relative charge; Multimerization.

Datasheet & SDS

Biological Description

Specificity
Phospho-Sin1 (Thr86) Antibody [L3G17] detects endogenous levels of total Sin1 protein only when it is phosphorylated at Thr86.

Clone
L3G17

Synonym(s)
JC310; MAPK associated protein 1; MAPKAP1; MEKK2-interacting protein 1; MGC2745; MIP1; SIN1; SIN1b; SIN1g; Target of rapamycin complex 2 subunit MAPKAP1; TORC2 subunit MAPKAP1

Background
Phosphorylation of Sin1 at threonine 86 (Thr86) represents a critical regulatory modification controlling the mechanistic target of rapamycin complex 2 (mTORC2), which comprises mTOR, rictor, Sin1, and mLST8 and functions as a key regulator of cell survival, growth, and cytoskeletal organization through phosphorylation of AGC kinase family members, including Akt at serine 473. Sin1 serves as an essential scaffolding component of mTORC2, and phosphorylation at Thr86 alongside Thr398 governs the integrity and catalytic activity of the entire mTORC2 complex through conformational changes that influence Sin1 association with other complex subunits. The functional consequences of Thr86 phosphorylation remain subject to cellular context, with two distinct regulatory paradigms emerging—Akt functions as the predominant kinase phosphorylating Sin1 at Thr86 across diverse cell lines and stimulation conditions, establishing a positive feedback loop wherein PDK1-mediated Akt phosphorylation at Thr308 generates partially active Akt that subsequently phosphorylates Sin1 at Thr86, enhancing mTORC2 kinase activity and enabling full Akt activation through mTORC2-mediated phosphorylation at Ser473. Conversely, phosphorylation of Sin1 at Thr86 and Thr398 by either S6K downstream of mTORC1 or by Akt itself triggers negative regulation by inducing Sin1 dissociation from the mTORC2 complex, thereby suppressing mTORC2 kinase activity and inhibiting Akt phosphorylation at Ser473 in response to insulin, IGF-1, PDGF, and EGF stimulation, establishing a feedback inhibition mechanism distinct from canonical IRS-1 and Grb10-mediated pathways. This dual regulatory capacity positions Thr86 phosphorylation as a molecular switch balancing mTORC2 activation and suppression depending on upstream signaling intensity, nutrient availability, and growth factor context. The phosphorylation state of Sin1 at Thr86 modulates mTORC2-dependent phosphorylation of additional substrates, including SGK1 at Ser422 and PKCα at Ser657, extending regulatory control beyond Akt to influence ion transport, cell migration, and membrane trafficking processes. Sin1 Thr86 phosphorylation integrates signals from both mTORC1 and growth factor receptor pathways, coordinating cellular responses to nutrient status and mitogenic stimulation through reciprocal crosstalk between mTORC1-S6K and mTORC2-Akt signaling axes. Cancer-associated mutations proximal to the Thr86 phosphorylation site, exemplified by the Sin1-R81T mutation identified from patient samples, impair phosphorylation-dependent negative regulation by preventing efficient phosphorylation, resulting in constitutive mTORC2 hyperactivation and sustained Akt signaling that drives oncogenic transformation, proliferation, and survival.

Background

Phosphorylation of Sin1 at threonine 86 (Thr86) represents a critical regulatory modification controlling the mechanistic target of rapamycin complex 2 (mTORC2), which comprises mTOR, rictor, Sin1, and mLST8 and functions as a key regulator of cell survival, growth, and cytoskeletal organization through phosphorylation of AGC kinase family members, including Akt at serine 473. Sin1 serves as an essential scaffolding component of mTORC2, and phosphorylation at Thr86 alongside Thr398 governs the integrity and catalytic activity of the entire mTORC2 complex through conformational changes that influence Sin1 association with other complex subunits. The functional consequences of Thr86 phosphorylation remain subject to cellular context, with two distinct regulatory paradigms emerging—Akt functions as the predominant kinase phosphorylating Sin1 at Thr86 across diverse cell lines and stimulation conditions, establishing a positive feedback loop wherein PDK1-mediated Akt phosphorylation at Thr308 generates partially active Akt that subsequently phosphorylates Sin1 at Thr86, enhancing mTORC2 kinase activity and enabling full Akt activation through mTORC2-mediated phosphorylation at Ser473. Conversely, phosphorylation of Sin1 at Thr86 and Thr398 by either S6K downstream of mTORC1 or by Akt itself triggers negative regulation by inducing Sin1 dissociation from the mTORC2 complex, thereby suppressing mTORC2 kinase activity and inhibiting Akt phosphorylation at Ser473 in response to insulin, IGF-1, PDGF, and EGF stimulation, establishing a feedback inhibition mechanism distinct from canonical IRS-1 and Grb10-mediated pathways. This dual regulatory capacity positions Thr86 phosphorylation as a molecular switch balancing mTORC2 activation and suppression depending on upstream signaling intensity, nutrient availability, and growth factor context. The phosphorylation state of Sin1 at Thr86 modulates mTORC2-dependent phosphorylation of additional substrates, including SGK1 at Ser422 and PKCα at Ser657, extending regulatory control beyond Akt to influence ion transport, cell migration, and membrane trafficking processes. Sin1 Thr86 phosphorylation integrates signals from both mTORC1 and growth factor receptor pathways, coordinating cellular responses to nutrient status and mitogenic stimulation through reciprocal crosstalk between mTORC1-S6K and mTORC2-Akt signaling axes. Cancer-associated mutations proximal to the Thr86 phosphorylation site, exemplified by the Sin1-R81T mutation identified from patient samples, impair phosphorylation-dependent negative regulation by preventing efficient phosphorylation, resulting in constitutive mTORC2 hyperactivation and sustained Akt signaling that drives oncogenic transformation, proliferation, and survival.

References
https://pubmed.ncbi.nlm.nih.gov/24161930/ https://pubmed.ncbi.nlm.nih.gov/26235620/

Protein Size (kDa)	PVDF Transfer Membrane Pore Size (μm)
< 10	0.22
10-20	0.22
20-30	0.45
30-45	0.45
45-70	0.45
80-100	0.45
100-140	0.45
> 140	0.45

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Protein Size (kDa)	Separating Gel Percentage
4-40	20%
12-45	15%
10-70	12.5%
15-100	10%
25-100	8%
60-210	5%