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| Specific Observation(s) |
Source of Data |
| S1P causes a small but significant increase in phosphoAKT |
RAW Ligand Screen: Phosphoprotein
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| S1P decreases LPA-induced AKT response |
RAW 2-Ligand Screen: Phosphoprotein
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| S1P decreases PAF-induced AKT response |
RAW 2-Ligand Screen: Phosphoprotein
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| S1P decreases MCF-induced AKT response |
RAW 2-Ligand Screen: Phosphoprotein
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| Abstract |
| In addition to its ability to induce a small but reproducible activation of the PI3K/AKT pathway, sphingosine-1-phosphate may also be activating a pathway for degradation of PI-3,4,5-P3 or dephosphorylation of AKT. |
| Sphingosine-1-phosphate (presumably by acting through an Edg family GPCR and probably via Gi and beta/gamma activation of class Ib PI3K) causes a small but significant increase in phosphoAKT that peaks at one minute and is back to baseline at 5 minutes after stimulation (phosphoprotein database). However, when sphingosine-1-phosphate is added in combination with another PI3K/AKT activator (e.g. lysophosphatidic acid, platelet activating factor or macrophage stimulating factor), the phosphoAKT response is smaller than that of the other factor alone. Lysophosphatidic acid and platelet activating factor, like sphingosine-1-phosphate act through GPCRs and presumably activate class Ib PI3K. However, macrophage colony stimulating factor acts through a receptor protein-Tyr kinase to activate class 1a PI3K. The fact that two different pathways for AKT activation are both inhibited by sphingosine-1-phosphate suggests that sphingosine-1-phosphate is inhibiting downstream of PI3K by either stimulating a phosphatase that degrades PI-3,4,5-P3 or a phosphatase that dephosphorylates AKT.
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| The possibility that sphingosine-1-phosphate stimulates a PI-3,4,5-P3 phosphatase is attractive for two reasons. First, it explains why the duration of AKT activation is very short when sphingosine-1-phosphate alone is added to RAW 264.7 cells. Secondly, it is consistent with sphingosine-1-phosphate stimulating chemotaxis, as has been observed in several cell types. Chemotaxis often involves activation of PI3K at the leading edge of the cell and in some cells this is followed by activation of PTEN (a PI-3,4,5-P3 phosphatase) in the trailing end of the cell (observed in D discoidum but controversial in neutrophils). This sharpens the PI-3,4,5-P3 gradient from the front to the back of the cells and promotes directional growth. |
| Suggested Future Experiments |
A knockdown of PTEN would be expected to eliminate the negative effect of sphingosine-1-phosphate on lysophosphatidic acid, platelet activating factor and macrophage colony stimulating factor dependent activation of AKT. PTEN knockdown would also be predicted to enhance activation of AKT by sphingosine-1-phosphate as a single ligand. The problem is that feedback loops due to loss of PTEN over a long time are likely to complicate the experiment.
Curiously, sphingosine-1-phosphate did not appear to reduce the AKT activation by C5a. This may be because C5a (at concentrations used) produces so much PI-3,4,5-P3 that it is in large excess over that needed to maximally stimulate AKT. Lower concentrations of C5a may reveal a negative effect of sphingosine-1-phosphate on AKT activation. |
| Discussion |
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[07 APR 2005]
Heping Han,
Univ. Texas Southwestern Medical Center
- In addition to the plausible hypotheses regarding roles of phosphatases, we suggest another one to explain the less than additive effects of MCF and SIP on phosphorylation of Akt.
- MCF induces tyrosine phosphorylation of c-Cbl and its subsequent association with Crk-II and the p85 subunit of PI3-kinase (Oncogene 14: 2331-8, 1997).
- Binding activity of c-Crk II is apparently regulated by phosphorylation of tyrosine 221; phosphorylation reduces binding to complexes (AfCS mini molecule page for Crk, EMBO J. 13:2341-51, 1994).
- S1P promotes Tyr phosphorylation of Crk (JBC 272: 16211-5, 1997), and thus dissociation of Crk from the MCF-stimulated signaling complex
- If Crk association is required for MCF activation of PI3 kinase and phosphorylation of Akt, then Crk may be a link between the SIP and MCF signaling pathways. This hypothesis could be tested by over-expressing a mutant form of Crk with Tyr 221 substituted by phenylalanine to prevent phosphorylation. If our hypothesis is correct then we would expect to reduce the SIP effect on MCF-stimulated phosphorylation of Akt.
Heping Han and Susanne Mumby
AfCS Antibody Lab
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