SUNDAY, DECEMBER 4- Late Abstracts 1 - Molecular Biology of the ...

SUNDAY
either heregulin or forskolin. The molecular mechanisms that mediate the proliferation of
Schwann cells in response to heregulin and forskolin are not well known. This study was
undertaken to identify the role of the cyclic AMP protein kinase A signaling pathway in Schwann
cells that are cultured with heregulin and forskolin. Studies conducted previously have shown
that the A-Kinase anchoring proteins of the cAMP/PKA pathway play an important role in
Schwann cell division. Incubation of neonatal rat Schwann cells with SiRNA oligos synthesized
against AKAP95 and AKAP150 resulted in a reduction in protein levels of both AKAPs
accompanied by a decrease in cell proliferation. In addition to a decrease in AKAP protein
levels, Schwann cells cultured with the SiRNA oligos against AKAP95 and AKAP150, exhibited
a reduction in protein levels of cyclin D3 and PKB/Akt respectively. To investigate if the loss of
AKAPs along with cyclin D3 occurred at the level of synthesis or degradation, neonatal rat
Schwann cells were cultured with the protein synthesis inhibitor cycloheximide and the
proteasome inhibitor lactacystin. Immunoblot analysis of Schwann cells incubated with
cycloheximide and SiRNA oligos or cycloheximide alone showed a reduction in both AKAP95
and cyclin D3 levels. Schwann cells cultured with lactacystin and SiRNA oligos showed a
marked reduction in AKAP95 and cyclin D3 levels in comparison to cultures incubated with
lactacystin only. These preliminary observations suggests that the loss of AKAP95 along with
the cyclin D3 proteins does not occur at the synthesis or degradation levels and instead may be
mediated by a different mechanism. To examine if AKAP150 associates with PKB/Akt to
stimulate Schwann cell proliferation, cells were incubated with modified N2 media or modified N2
media with heregulin and forskolin. Using immunofluorescent staining techniques, preliminary
results demonstrate that Schwann cells treated with heregulin and forskolin exhibit distinct
colocalization patterns for AKAP150 and PKB/Akt. These results suggest that in the presence of
forskolin and heregulin, AKAP150 may associate with PKB/Akt to mediate cell proliferation.
2029
Turn motif phosphorylation negatively regulates activation-loop phosphorylation in Akt.
D. Hiraoka 1 , E. Okumura 1 , T. Kishimoto 1 ; 1 Tokyo Institute of Technology, Yokohama,
Kanagawa, Japan
Akt, also known as PKB, plays a central role in various signaling pathways that regulate cellular
processes such as metabolism, proliferation and survival. Under stimulation, phosphorylation of
the activation loop (A-loop) and hydrophobic motif (HM) of Akt by the kinase, PDK1, and the
mammalian target of rapamycin complex 2 (mTORC2), respectively, results in its activation. A
well-conserved threonine in the turn motif (TM) is also constitutively phosphorylated by
mTORC2 and contributes to the stability of Akt. However, the relationship between TM
phosphorylation and the phosphorylation of Akt on the HM and A-loop has not been sufficiently
evaluated. Using starfish oocytes as a model system, our study provides the first evidence that
TM phosphorylation plays a negative role in A-loop phosphorylation. In this system, the
maturation-inducing hormone, 1-methyladenine, stimulates Akt to induce meiotic reinitiation
through the activation of cyclin B-Cdc2. The phosphorylation status of Akt was monitored via
introduction of exogenous human Akt in starfish oocytes. Injection of an anti-starfish TOR
antibody inhibited TM and HM phosphorylation, suggesting that phosphorylation of these sites
depends on TOR, as reported in mammalian cells. Precise analyses of single or double alanine
substitution mutants at each of three phosphorylation residues revealed that TM
phosphorylation renders Akt susceptible to dephosphorylation on the A-loop. When A-loop
phosphatase was inhibited by okadaic acid, TM phosphorylation still reduced A-loop
phosphorylation, suggesting that the effect is caused at least partially through reduction of
sensitivity to PDK1. By contrast, HM phosphorylation enhanced A-loop phosphorylation and
achieved full activation of Akt via a mechanism at least partially independent of TM