Korean J Physiol Pharmacol.  2011 Aug;15(4):245-249. 10.4196/kjpp.2011.15.4.245.

Amphetamine-induced ERM Proteins Phosphorylation Is through PKCbeta Activation in PC12 Cells

• Jeong HJ
• Kim JH
• Jeon S

• KMID: 1448929
• DOI: http://doi.org/10.4196/kjpp.2011.15.4.245

Abstract

Amphetamine, a synthetic psychostimulant, is transported by the dopamine transporter (DAT) to the cytosol and increases the exchange of extracellular amphetamine by intracellular dopamine. Recently, we reported that the phosphorylation levels of ezrin-radixin-moesin (ERM) proteins are regulated by psychostimulant drugs in the nucleus accumbens, a brain area important for drug addiction. However, the significance of ERM proteins phosphorylation in response to drugs of abuse has not been fully investigated. In this study, using PC12 cells as an in vitro cell model, we showed that amphetamine increases ERM proteins phosphorylation and protein kinase C (PKC) beta inhibitor, but not extracellular signal-regulated kinase (ERK) or phosphatidylinositol 3-kinases (PI3K) inhibitors, abolished this effect. Further, we observed that DAT inhibitor suppressed amphetamine-induced ERM proteins phosphorylation in PC12 cells. These results suggest that PKCbeta-induced DAT regulation may be involved in amphetmaine-induced ERM proteins phosphorylation.

Keyword

Amphetamine; ERM proteins (ezrin, radixin, moesin); PKCbeta; PC12 cells; Dopamine transporter

MeSH Terms

Amphetamine
Animals
Brain
Cytosol
Dopamine
Dopamine Plasma Membrane Transport Proteins
Nucleus Accumbens
PC12 Cells
Phosphatidylinositol 3-Kinases
Phosphorylation
Phosphotransferases
Protein Kinase C
Proteins
Street Drugs
Substance-Related Disorders
Amphetamine
Dopamine
Dopamine Plasma Membrane Transport Proteins
Phosphatidylinositol 3-Kinases
Phosphotransferases
Protein Kinase C
Proteins
Street Drugs

Figure

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  Fig. 1. Amphetamine induces ERM proteins phosphorylation in PC12 cells. Serum reduced PC12 cells were treated with 5μM of AMPH for the indicated time, and whole cell lysates were immunoblotted with anti-p-ERM or ERM antibody. The intensity of the phosphorylated ERM band was normalized to that of total ERM. The data represent the means±SE of three independent experiments. ∗p<0.05 vs. C.

• 

  Fig. 2. Amphetamine-induced ERM proteins phosphorylation is not mediated by Akt or ERK activation in PC12 cells. (A) Serum reduced PC12 cells were treated with 5μM of AMPH for the indicated time, and whole cell lysates were immunoblotted with anti-p-Akt or Akt antibody and (B) anti-p-ERK or ERK antibody. (C) Serum reduced PC12 cells were pre-incubated with 30μM of LY294002 or 10μM of PD098059 for 30 min, and then treated with AMPH for 4 h. The whole cell lysates were immunoblotted with anti-p-ERM or ERM antibody. The intensity of the phosphorylated ERM, Akt and ERK band was normalized to that of total ERM, Akt, and ERK, respectively. The data represent the means±SE of three independent experiments. ∗p<0.05 vs. C.C, unstimulated PC12 cells; A, AMPH-stimulated cells.

• 

  Fig. 3. Amphetamine-induced ERM proteins phosphorylation is related with PKC β and DAT activation in PC12 cells. (A) Serum reduced PC12 cells were treated with 5 μM of AMPH for the indicated time, and whole cell lysates were immunoblotted with anti-p-PKCβ or PKCβ antibody. (B) PC12 cells were pre-incubated with 10 μM of GBR12909 (DAT I) or 0.5 μM of Enzastaurin (PKCβ I) for 30 min, and then treated with 5 μM of AMPH for 4 h, and whole cell lysates were immunoblotted with indicated antibodies. The intensity of the phosphorylated PKCβ and ERM band was normalized to that of total ERM and PKCβ. The data represent the means±SE of three independent experiments. ∗p<0.05 vs. C, C, unstimulated PC12 cells; A, AMPH-stimulated cells.

Reference

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