We have identified an unusual potential dual Akt/protein kinase B consensus phosphorylation motif in the protein Synip (RxKxRS97xS99). Surprisingly, serine 97 is not appreciably phosphorylated, 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 dissociation. Furthermore, overexpression of WT-Synip in 3T3L1 adipocytes had no effect on insulin-stimulated recruitment of glucose transporter 4 (GLUT4) to the plasma membrane, 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 Synip–Syntaxin4 interaction.

To determine the downstream signaling pathways regulated by betacellulin (BTC) in comparison with epidermal growth factor (EGF), we used Chinese hamster ovary cells overexpressing the human EGF receptor (ErbB1/EGFR). The overall time-dependent activation of EGFR autophosphorylation was identical in cells treated with 1 nm BTC or 1.5 nm EGF. Analysis of site-specific EGFR phosphorylation demonstrated that the BTC and EGF tyrosine phosphorylation of Y1086 was not significantly different. In contrast, the autophosphorylation of Y1173 was markedly reduced in BTC-stimulated cells, compared with EGF stimulation that directly correlated with a reduced BTC stimulation of Shc tyrosine phosphorylation, Ras, and Raf-1 activation. On the other hand, Y1068 phosphorylation was significantly increased after BTC stimulation, compared with EGF in parallel with a greater extent of Erk phosphorylation. Expression of a dominant interfering MEK kinase 1 (MEKK1) and Y1068F EGFR more efficiently blocked the enhanced Erk activation by BTC, compared with EGF. Interestingly BTC had a greater inhibitory effect on apoptosis, compared with EGF, and expression of Y1068F EGFR abolished this enhanced inhibitory effect. Together, these data indicated that although BTC and EGF share overlapping signaling properties, the ability of BTC to enhance Erk activation occurs independent of Ras. The increased BTC activation results from a greater extent of Y1068 EGFR tyrosine phosphorylation and subsequent increased recruitment of the Grb2-MEKK1 complex to the plasma membrane, compared with EGF stimulation. The increased Erk activation by BTC associated with antiapoptotic function.

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Insulin stimulation results in the activation of cyclin-dependent kinase-5 (CDK5) in lipid raft domains via a Fyn-dependent phosphorylation on tyrosine residue 15. In turn, activated CDK5 phosphorylates the Rho family GTP-binding protein TC10␣ on threonine 197 that is sensitive to the CDK5 inhibitor olomoucine and blocked by small interfering RNA-mediated knockdown of CDK5. The phosphorylation deficient mutant T197A-TC10␣ was not phosphorylated and excluded from the lipid raft domain, whereas the phosphorylation mimetic mutant (T197D-TC10␣) was lipid raft localized. Insulin resulted in the GTP loading of T197D-TC10␣ but not T197A-TC10␣ and in parallel, T197D-TC10␣ but not T197A-TC10␣ depolymerized cortical actin and inhibited insulin-stimulated GLUT4 translocation. These data demonstrate that CDK5-dependent phosphorylation maintains TC10␣ in lipid raft compartments thereby disrupting cortical actin, whereas subsequent dephosphorylation of TC10␣ through inactivation of CDK5 allows for the re-assembly of F-actin. Because cortical actin reorganization is required for insulin-stimulated GLUT4 translocation, these data are consistent with a CDK5-dependent TC10␣ cycling between lipid raft and non-lipid raft compartments.Similar to fibroblasts, pre-differentiated adipocytes are elongated and relatively flat cells that contain well defined actin stress fibers. In contrast, following differentiation into mature adipocytes the cells become highly spherical with a relatively thick cortical actin lining the inner surface of the plasma membrane. This cortical actin structure in adipocytes is composed of patches of punctate F-actin that emanates from organized caveolae-rosettes containing the caveolin protein refered to as Cav-actin (1). This cortical organization of F-actin is dependent on caveolae-rosette organization as disruption of caveolae results in the dissolution of cortical actin, whereas depolymerization of cortical actin has no effect on caveolae-rosette organization (2, 3). Although insulin induces the rapid breakdown of stress fibers and the appearance of lamellipodia in pre-adipocytes, in differentiated 3T3L1 adipocytes and primary rat adipocytes insulin primarily results in dynamic actin rearrangements in both the cortical and perinuclear regions (4, 5).Caveolin-enriched domains in adipocytes have also been reported to associate with a variety of structural and functional proteins. For example, the structural protein flotillin is lipid raft localized and interacts with CAP, Cbl, and Fyn (6). In particular, the unusual Rho family member GTP-binding protein TC10 appears also to be lipid raft localized in adipocytes and has been implicated in the regulation of insulin-stimulated GLUT4 translocation through the assembly of membrane docking regulating proteins (7). In parallel, TC10 appears to differentially regulate two distinct compartmentalized actin populations. Overexpression of TC10 was found to disrupt cortical actin when lipid raft localized through its endogenous carboxyl-terminal domain that specifies bot...