SHIP1 is mainly expressed in hematopoietic cells and appears to regulate signaling for cell growth, differentiation, apoptosis, and FcγRIIB-mediated inhibitory signals in hematopoietic cells (14
). Although SHIP1 is a relatively hematopoietic cell-specific phosphoinositol 5′-phosphatase, previous reports have also indicated the possible involvement of its 5′-phosphatase activity in insulin signaling. In this regard, insulin-induced Xenopus
oocyte maturation and Glut4 translocation in 3T3-L1 adipocytes were blocked by exogenous expression of WT-SHIP1 (15
). Because the expression of SHIP1 is negligible in insulin's target tissues (38
), it was predicted that an alternative molecule capable of regulating insulin-induced generation of PI(3,4,5)P3 by an intrinsic phosphoinositide 5′-phosphatase activity must exist. Along this line, Pesesse et al. and we recently cloned a SHIP1 isozyme, SHIP2, which is abundantly expressed in insulin's target tissues including skeletal muscle and fat cells (22
). Although SHIP1 and SHIP2 have structural similarities, the substrate specificities of these SHIP family members were found to be somewhat different. SHIP2 selectively hydrolyzes PI(3,4,5)P3, whereas SHIP1 hydrolyzes both PI(3,4,5)P3 and inositol 1,3,4,5-tetraphosphate in vitro (56
). Therefore, our results with SHIP2 appear to strengthen the case for a physiological role for its 5′-phosphatase activity specifically toward PI(3,4,5)P3, which plays a key role in insulin signaling.
SHIP1 is shown to possess in vivo 5′-phosphatase activity, which was clearly demonstrated by the fact that interleukin-3-stimulated macrophages derived from SHIP1 knockout mice revealed a higher content of PI(3,4,5)P3 than those from control littermates (21
). Because SHIP2 was cloned based on the homology of the conserved catalytic region among the already-known 5′-phosphatases (22
), SHIP2 was also postulated to possess 5′-phosphatase activity. In fact, SHIP2 has already been reported to possess 5′-phosphatase activity in vitro (19
). However, the ability of SHIP2 catalytic activity to regulate phosphoinositides in vivo was uncertain. Our results here clearly demonstrate that overexpression of WT-SHIP2 decreased insulin-induced generation of PI(3,4,5)P3, with a concomitant increase in the amount of PI(3,4)P2 in 3T3-L1 adipocytes (Fig. ). The modulation of the amount of phosphoinositides was not elicited by the involvement of SHIP2 in an early part of the insulin signaling pathway, up to activation of PI 3-kinase, because insulin-induced tyrosine phosphorylation of the insulin receptor β subunit and IRS-1, IRS-1 association of the p85 subunit of PI 3-kinase, and PI 3-kinase activity were not affected by expression of either WT-SHIP2 or ΔIP-SHIP2 (Fig. ). Taken together, the results of the present study indicate that SHIP2 can modulate insulin signaling in vivo by specifically hydrolyzing PI 3-kinase products via its 5′-phosphatase activity. Importantly, expression of catalytically inactive SHIP2, ΔIP-SHIP2, increased PI(3,4,5)P3 content even in the basal state, and greater production of PI(3,4,5)P3 was elicited by insulin treatment than was elicited in control LacZ-transfected cells. These results indicate that ΔIP-SHIP2 functions in a dominant-negative manner toward endogenous SHIP2 by possibly competing with the generated phosphoinositides.
PI 3-kinase is recognized as the critical molecule for mediating the various metabolic actions of insulin (16
). The key phosphoinositide that functions as the lipid second messenger is thought to be PI(3,4,5)P3 generated from PI(4,5)P2 by activated PI 3-kinase (17
). It is also known that insulin stimulation increases the cellular concentrations of PI(3,4)P2 and PI(3,4,5)P3 (17
). The molecular events downstream of PI 3-kinase possibly regulated by these phospholipids, which lead to glucose transport, have been extensively studied. With respect to Akt, there are previous reports indicating both positive and negative roles in insulin-induced glucose transport. For example, overexpression of the constitutively active form of Akt increased glucose uptake in 3T3-L1 adipocytes (10
) and the expression of a dominant-negative form of Akt inhibited insulin-induced glucose uptake in L6 myoblasts (41
). In contrast, Kitamura et al. argued against a physiological role for Akt in insulin-induced glucose in 3T3-L1 adipocytes (25
). Although there is still controversy on the physiological relevance of Akt in glucose transport, it is important to clarify how PI(3,4)P2 and PI(3,4,5)P3 are involved in the activation of Akt. Akt becomes active by its phosphorylation on both Thr308
residues by PDK1 and an unknown kinase tentatively referred to as PDK2, respectively (2
). Previous in vitro studies suggest that Akt is activated by PI(3,4)P2 but not by PI(3,4,5)P3 in the absence of PDK1 (16
). In contrast, in the presence of PDK1, Akt activation was preferentially induced by PI(3,4,5)P3 rather than by PI(3,4)P2 (1
). Thus, the roles of PI(3,4,5)P3 and PI(3,4)P2 in the activation of Akt in vitro remain unclear. The present study demonstrated that insulin-induced Akt activation by its phosphorylation on both Thr308
residues was inhibited by overexpression of WT-SHIP2 and enhanced by expression of ΔIP-SHIP2 (Fig. and data not shown). Because SHIP2 possesses in vivo 5′-phosphatase activity, SHIP2 appears to negatively regulate insulin-induced Akt activation by hydrolyzing PI(3,4,5)P3 to PI(3,4)P2. Our results do not completely rule out the involvement of PI(3,4)P2 in this activation process. Along this line, both PI(3,4,5)P3 and PI(3,4)P2 bind with high affinity to the pleckstrin homology domain of Akt. This leads to the recruitment of Akt to the plasma membrane to be phosphorylated (1
). However, it is logical to propose that PI(3,4,5)P3 has a greater role than PI(3,4)P2 in insulin-induced Akt activation. This idea is supported by recent reports showing that mast cells and B lymphocytes derived from SHIP1-deficient mice exhibited enhanced Akt activation following ligand stimulations (3
) and that reduction of endogenous SHIP2 protein expression by an antisense oligonucleotide approach resulted in increased Akt activity in HeLa cells (51
). PI(3,4,5)P3 could also be metabolized by a phosphatase and tensin homolog, deleted on chromosome 10 (PTEN), that is known to possess 3′-phosphoinositol phosphatase activity toward PI(3,4,5)P3 (32
). Studies with the cells derived from PTEN-deficient mice showed increased PI(3,4,5)P3 content and elevated Akt activity (44
). In addition, overexpression of wild-type PTEN inhibited insulin-induced Akt activity in 3T3-L1 adipocytes (34
). On the basis of these results, PI(3,4,5)P3 appears to be a key mediator for the activation of Akt in vivo.
Atypical isoforms of PKC (PKCζ and PKCλ) have been implicated as downstream effectors of PI 3-kinase involved in insulin-induced glucose uptake (27
). Original studies indicated that PI(3,4,5)P3 activated PKCζ more efficiently than PI(3,4)P2 (33
). However, a recent report indicated that both PI(3,4,5)P3 and PI(3,4)P2 were equally capable of stimulating PKCζ activity (46
). Thus, results from these previous in vitro studies are not consistent with the roles of PI(3,4,5)P3 and PI(3,4)P in the activation of atypical PKC. In addition, in vivo regulation of atypical PKC by these phospholipids has not been examined. In the present study, the role of SHIP2 in insulin-induced activation of PKCλ was examined, since PKCλ is robustly expressed in 3T3-L1 adipocytes (27
). Our results demonstrated that insulin-induced stimulation of PKCλ activity was markedly inhibited by WT-SHIP2 overexpression and significantly enhanced by expression of ΔIP-SHIP2. These results indicate that SHIP2 negatively regulates insulin-induced PKCλ activation via its 5′-phosphatase activity and that PI(3,4,5)P3 is more important than PI(3,4)P2 for this in vivo activation. Possible differences in the regulation of PKCλ and PKCζ by these phospholipids require further clarification.
Our results further clarified the functional localization of SHIP2 in the insulin signaling cascade. In this regard, activation of Akt and PKCλ induced by the constitutively active form of PI 3-kinase (Myr-p110) was inhibited by coexpression of WT-SHIP2 in an MOI-dependent manner (Fig. and ). In contrast, the activity of Akt and PKCλ induced by the expression of the constitutively active forms of Akt (Myr-Akt) and PKCλ (ΔPD-PKCλ) was not affected by coexpression of WT-SHIP2. On the basis of these results and the fact that SHIP2 does not affect insulin signaling up to the PI 3-kinase activation step, it is logical to conclude that SHIP2 functions, via its 5′-phosphatase activity, at a site distal to PI 3-kinase, and proximal to Akt and PKCλ, of the insulin signaling system in 3T3-L1 adipocytes.
Because insulin-stimulated activation of both Akt and PKCλ was negatively regulated by SHIP2, one can speculate that SHIP2 is involved in the regulation of insulin-induced glucose transport. Importantly, our results demonstrated that both insulin-stimulated 2-DOG uptake and Glut4 translocation were effectively inhibited by expression of WT-SHIP2 and enhanced by expression of ΔIP-SHIP2 (Fig. ). These results indicate the involvement of SHIP2 in insulin stimulation of glucose transport via the 5′-phosphatase activity in 3T3-L1 adipocytes. Our results are consistent with a previous SHIP1 study showing that exogenous expression of WT-SHIP1 also inhibited insulin-induced Glut4 translocation in 3T3-L1 adipocytes (54
). On the other hand, our results showed that ΔIP-SHIP2 expression enhanced insulin-induced Glut4 translocation, whereas no apparent effect of the expression of a 5′-phosphatase-defective SHIP1 was seen in the previous report (54
). The reason for this difference is uncertain. However, we suggest the following possibilities. First, this may arise from a methodological difference between the analyses. In this regard, we expressed SHIP2 by utilizing adenovirus-mediated gene transfer, while nuclear microinjection was employed to express SHIP1 in the previous study. Second, the phosphatase-defective SHIP1 may not inhibit the function of endogenous SHIP2, because SHIP1 is not in fact the SHIP family protein member expressed in 3T3-L1 adipocytes. This hypothesis was supported by the recent report indicating different substrate specificities for SHIP1 and SHIP2 (56
). Third, our ΔIP-SHIP2 was constructed by mutating three amino acids conserved within the 5′-phosphatase region, whereas only one mutation was introduced into the mutant SHIP1 in the previous report (54
). We cannot precisely determine the possible difference in the remaining 5′-phosphatase activity between the two mutants, because the previous study did not measure the amounts of phosphoinositides generated in vivo. However, it is possible to speculate that the 5′-phosphatase activity in ΔIP-SHIP2 was more profoundly defective than that in the mutant SHIP1 in the previous study. In any case, our results with SHIP2 clearly indicate the physiological impact of the 5′-phosphatase activity on insulin-induced glucose transport in 3T3-L1 adipocytes, although the impact of SHIP2 on glucose metabolism in the whole body awaits further investigation with knockout mice.
2-DOG uptake and Glut4 translocation in the basal states were significantly greater in ΔIP-SHIP2-transfected cells than in LacZ-transfected control 3T3-L1 adipocytes. We assume that these increases are not nonspecific effects, because transfection with either control LacZ itself or WT-SHIP2 did not change the basal values of 2-DOG uptake and Glut4 translocation. Although basal Akt activity was not increased, basal PKCλ activity had a tendency, although not a statistically significant one, to increase in ΔIP-SHIP2 cells. In addition, Akt phosphorylation, detected by utilizing the antiphosphospecies-specific Ser473 Akt antibody, also tended to increase (data not shown). It is of note that basal amounts of PI(3,4,5)P3 were increased by expression of ΔIP-SHIP2, possibly caused by inhibition of the basal function of endogenous SHIP2 by expression of ΔIP-SHIP2. Basal elevation of PI(3,4,5)P3 amounts may lead to increased effects of the downstream events, although this is speculative. Alternatively, measurements of basal 2-DOG uptake and Glut4 translocation may be more sensitive, at least in our experimental conditions, than those of Akt and PKCλ activities in response to the elevation of PI(3,4,5)P3 levels.
Another important metabolic action of insulin is to stimulate glycogen synthesis. Activation of glycogen synthase is the key step in insulin-induced glycogen synthesis (5
). Previous reports indicate two possible dephosphorylation mechanisms for activating glycogen synthase. The activated Akt phosphorylates GSK3β, resulting in inactivation of GSK3β (11
). Since GSK3β phosphorylates glycogen synthase, inactivation of GSK3β by Akt leads to the activation of glycogen synthase by preventing its phosphorylation (11
). Alternatively, glycogen synthase could be activated via PP1. Insulin-induced activation of PP1 dephosphorylates glycogen synthase, resulting in the activation of glycogen synthase (5
). Originally, studies suggested that PP1 might be regulated by a mitogen-activated protein (MAP) kinase cascade, because ribosomal S6 kinase 2, a downstream substrate for MAP kinase, could phosphorylate PP1 in vitro (28
). However, inhibition of the MAP kinase pathway by utilizing a pharmacological inhibitor did not affect insulin-induced PP1 activity or glycogen synthesis (29
). Subsequently, insulin-induced PP1 activation was found to be mediated by a PI 3-kinase dependent pathway (5
). In this regard, inhibition of PI 3-kinase activity by wortmannin inhibited insulin-induced PP1 activity (5
), although the precise signaling mechanisms by which PI 3-kinase activates PP1 are unknown.
The relative importance of GSK3β versus PP1 in insulin-induced glycogen synthase activation appears to be dependent on the cell types used for analysis. GSK3β is considered to be the key molecule in regulation of insulin-induced glycogen synthase in skeletal muscle cell lines such as L6 myotubes (52
). However, the role of GSK3β in 3T3-L1 adipocytes is unclear. A previous report suggested the involvement of GSK3β, because glycogen synthase activity was inhibited by overexpression of GSK3β in 3T3-L1 adipocytes (50
). In contrast, Ueki et al. argued against a physiological role for GSK3β in glycogen synthase activation in 3T3-L1 adipocytes (52
). A recent report emphasizes a switch from a role for GSK3β to a role for PP1 in the activation of glycogen synthase during differentiation into 3T3-L1 adipocytes (5
). Regardless of the relative importance of GSK3β versus PP1 in the regulation of the activation of glycogen synthase in 3T3-L1 adipocytes, overexpression of WT-SHIP2 inhibited both insulin-induced GSK3β phosphorylation and PP1 activation. Conversely, these signaling events were enhanced by expression of ΔIP-SHIP2 (Fig. and ). As the result, insulin-induced activation of glycogen synthase and glycogen synthesis were inhibited by overexpression of WT-SHIP2 and enhanced by expression of ΔIP-SHIP2 (Fig. and ). Therefore, our results indicate that SHIP2, via its 5′-phosphatase activity, is physiologically involved also in the regulation of the insulin signal leading to glycogen synthesis.
By overexpression of WT-SHIP2, insulin-induced activation of Akt, PKCλ, and PP1 and phosphorylation of GSK3β were partly inhibited (by 44.5, 50.0, 78.8, and 33.0%, respectively), whereas inhibition of PI 3-kinase activity by pharmacological inhibitors or expression of a dominant-negative form of PI 3-kinase was reported to elicit greater effects (7
). The reason why WT-SHIP2 overexpression only partially inhibited insulin-stimulated activation of the downstream effectors of PI 3-kinase is uncertain. One possible explanation is that the expression of WT-SHIP2 is not high enough to completely inhibit insulin's effects. Greater amounts of WT-SHIP2 expression (more than threefold greater than that of endogenous SHIP2) could not be obtained in our hands by utilizing an adenovirus-mediated expression system at an adequate MOI for the experiments. These technical difficulties appear to arise from an abundance of endogenous SHIP2 in 3T3-L1 adipocytes in addition to the relatively high molecular mass of SHIP2 (140 kDa). Another possible explanation is that there is a redundant pathway that regulates PI 3-kinase products, PI(3,4,5)P3. As mentioned above, PTEN is a possible candidate for mediating an alternative pathway for the hydrolysis of PI(3,4,5)P3 to PI(4,5)P2 in intact cells (32
). Interestingly, it is reported that overexpression of PTEN also resulted in a partial (~50%) inhibition of insulin-stimulated Akt activation in 3T3-L1 adipocytes (34
), although the physiological significance of PTEN has not been clarified. It would be interesting to further clarify how SHIP2 and PTEN may cooperatively or solely participate in a physiological down-regulation of PI(3,4,5)P3 generated by insulin.
Although SHIP2 is known to be tyrosine phosphorylated in response to insulin, the mechanisms by which SHIP2 might be activated during insulin signaling are unknown (19
). Based on the previous reports with SHIP1, tyrosine phosphorylation of SHIP1 in vitro by kinase Lck resulted in a two- to threefold reduction in the level of 5′-phosphatase activity (38
). In contrast, a recent report suggested that tyrosine phosphorylation of SHIP1 did not affect the total 5′-phosphatase activity of SHIP1 in B lymphocytes. Instead, the membrane localization of SHIP1 appeared to be important for hydrolyzing PI(3,4,5)P3 (40
). Along this line, it was more recently reported that tyrosine phosphorylation of SHIP2 in response to platelet-derived growth factor did not affect the phosphatase activity of SHIP2 in astrocytes (51
). Thus, future studies will be needed to clarify whether the tyrosine phosphorylation of SHIP2 affects its 5′-phosphatase activity or cellular localization in the insulin signaling system.
In summary, SHIP2 is abundantly expressed in insulin's target tissues including 3T3-L1 adipocytes. We clarified the role of SHIP2 in insulin signaling. SHIP2 has in vivo 5′-phosphatase activity capable of hydrolyzing PI(3,4,5)P3 to PI(3,4)P2. Via its 5′-phosphatase activity, SHIP2 was involved in insulin signaling at the level between activation of PI 3-kinase and of its effector molecules. The downstream molecules of PI 3-kinase including Akt, PKCλ, GSK3β, and PP1 all appeared to be preferentially activated by PI(3,4,5)P3 rather than by PI(3,4)P2. By regulating these effector molecules, SHIP2 appears to negatively regulate insulin-induced glucose uptake and glycogen synthesis in 3T3-L1 adipocytes. Since SHIP2 appears to be a negative regulator of insulin signaling, the increased enzymatic activity and/or the improper cellular SHIP2 localization might lead to inadequate hydrolysis of PI(3,4,5)P3 generated by insulin stimulation. This might be a part of the cause of insulin resistance seen in obesity and type 2 diabetes. Further studies would be required to investigate the possible involvement of SHIP2 in these disease states.