T H E J O U R N A L O F C E L L B I O L O G Y
The Journal of Cell Biology, Vol. 168, No. 6, March 14, 2005 921–928
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Akt2 phosphorylates Synip to regulate docking and
fusion of GLUT4-containing vesicles
and Masatomo Mori
Department of Medicine and Molecular Science, Gunma University Graduate School of Medicine, Maebashi, Gunma, 371-8511, Japan
Health and Science Center, Gunma University, Maebashi, Gunma, 371-8510, Japan
Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, Kawaguchi, Saitama 332-0012, Japan
e have identified an unusual potential dual
Akt/protein kinase B consensus phosphoryla-
tion motif in the protein Synip (RxKxRS
Surprisingly, serine 97 is not appreciably phosphory-
lated, whereas serine 99 is only a specific substrate for
Akt2 but not Akt1 or Akt3. Although wild-type Synip
(WT-Synip) undergoes an insulin-stimulated dissociation
from Syntaxin4, the Synip serine 99 to phenylalanine
mutant (S99F-Synip) is resistant to Akt2 phosphorylation
and fails to display insulin-stimulated Syntaxin4 dissocia-
tion. Furthermore, overexpression of WT-Synip in 3T3L1
adipocytes had no effect on insulin-stimulated recruitment
of glucose transporter 4 (GLUT4) to the plasma mem-
brane, whereas overexpression of S99F-Synip functioned
in a dominant-interfering manner by preventing insulin-
stimulated GLUT4 recruitment and plasma membrane
fusion. These data demonstrate that insulin activation
of Akt2 specifically regulates the docking/fusion step
of GLUT4-containing vesicles at the plasma membrane
through the regulation of Synip phosphorylation and
Akt, also known as protein kinase B (PKB), is an important
regulator of several cellular processes, including proliferation,
metabolism, and programmed cell death (Lawlor and Alessi,
2001; Whiteman et al., 2002). Akt has three isoforms, Akt1
), Akt2 (PKB
), and Akt3 (PKB
ping but distinct cellular function. In particular, recent studies
have demonstrated that Akt1 plays an important role on
growth and antiapoptosis, whereas Akt2 functions primarily as
a regulator of glucose metabolism (Cho et al., 2001b; Bae et
al., 2003). For example, Akt1
but relatively normal glucose homeostasis, whereas Akt2
mice displayed insulin-resistant glucose metabolism in liver
and muscle (Cho et al., 2001a). However, the insulin resis-
tance is relatively mild and becomes significantly more pro-
nounced in conjunction with the loss of Akt1 (Jiang et al.,
2003). Consistent with a primary role for Akt2, a family with
severe insulin resistance and overt diabetes was mapped to a
single point mutation in Akt2 (George et al., 2004). Thus,
there seems to exist intracellular signal specificity and some
), each with overlap-
mice have reduced body size
compensation mechanism for the regulation of glucose metab-
olism between Akt1 and Akt2.
It is well documented that Akt is involved in the regula-
tion of glucose metabolism by inhibiting glycogen synthesis
through the inhibition of glycogen synthesis kinase 3 (GSK3)
activity (Cross et al., 1995; Coghlan et al., 2000; Doble and
Woodgett, 2003). However, how Akt regulates glucose uptake
(muscle and adipose tissue), its association with peripheral
insulin resistance, and the molecular basis for the apparent Akt2
specificity is still unknown.
Peptide substrate mapping studies have identified the
preferred Akt1 phosphorylation consensus site as RxRxxS/T
(Alessi et al., 1996). Currently, over 20 substrates for Akt have
been identified; however, none of these substrates has been
reported to exhibit Akt isoform selectivity. Thus, at present, the
molecular basis for the physiologic specificities of Akt isoform
function remains a fundamental issue that has yet to be resolved.
In this regard, we previously identified Synip as a Syntaxin4
interacting protein (Min et al., 1999). Under the basal conditions,
Synip was constitutively bound to Syntaxin4 and prevented the
interaction of Syntaxin4 with both SNAP23 (synaptosome-
associated proteins of 23 kD) and VAMP2 (vesicle-associated
membrane protein 2; Min et al., 1999). Insulin treatment resulted
in a dissociation of the Synip–Syntaxin4 complex allowing for
Correspondence to Shuichi Okada: email@example.com
Abbreviations used in this paper: GLUT4, glucose transporter 4; GSK3, glycogen
synthesis kinase 3; PI3, phosphatidylinositol 3; siRNA, small interfering RNA;
JCB • VOLUME 168 • NUMBER 6 • 2005928
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