Rab5 regulates PI synthesis and turnover in the endocytic pathway
Rab5 regulates a complex network of effector proteins that are recruited on the early endosome membrane by binding to PtdIns(3)P (Simonsen et al., 1998
; Christoforidis et al., 1999b
; Nielsen et al., 2000
; Schnatwinkel et al., 2004
). PtdIns(3)P is an important molecular hallmark of the endocytic pathway (Wurmser and Emr, 1998
). It is required for early endosome fusion and motility along microtubules (Christoforidis et al., 1999a
; Hoepfner et al., 2005
), phagosome maturation (Vieira et al., 2001
), multivesicular body formation (Futter et al., 2001
), and signaling (Tsukazaki et al., 1998
). The findings that the lipid kinase that generates PtdIns(3)P, hVps34, is also a Rab5 effector (Christoforidis et al., 1999b
) and that the effectors form large oligomeric complexes on the early endosome membrane (McBride et al., 1999
), led us to propose that Rab5 regulates the formation of a membrane domain on the early endosome enriched in PtdIns(3)P and containing the various effector proteins required for early endosome tethering, fusion, and motility (Zerial and McBride, 2001
). Our present results strengthen this model with the demonstration that the synthesis of PtdIns(3)P is regulated by Rab5 itself.
Among the PIs tested, PtdIns(3)P was the major 3-PI present on early endosomes, despite the generation of other PI species on the plasma membrane and a continuous membrane flow toward the early endosomes. Furthermore, PtdIns(3)P is enriched in the Rab5 domain and less abundant or absent from other subcompartments of early and recycling (Rab4- and Rab11-positive) endosomes. In addition to hVps34, Rab5 interacts also with PI3Kβ, a type I PI 3-K involved in the generation of PtdIns(3,4,5)P3 and PtdIns(3,4)P2 upon stimulation at the plasma membrane. Even in the presence of the constitutively active Rab5Q79L mutant, PtdIns(3,4,5)P3 was localized to the plasma membrane but not early endosomes, suggesting that turnover of this PI must occur early to maintain the specificity of PtdIns(3)P localization in the endosomal system. The discovery that Rab5 interacts with, and stimulates the enzymatic activity of, 5- and 4-Pase, provides an explanation for how such synthesis and turnover can be coordinated.
We propose that the synthesis of PtdIns(3,4,5)P3 at the plasma membrane is coupled either to the dephosphorylation of the 3′ position by PTEN, thus leading to PtdIns(4,5)P2, or to the sequential dephosphorylation by 5- and 4-Pase, leading to PtdIns(3)P. Such enzymatic cascade could initiate at the plasma membrane, where under certain stimulatory conditions pools of PtdIns(3)P can be detected (Maffucci et al., 2003
), and continue along transport to the early endosomes, given that PI3Kβ is detected in clathrin-coated vesicles (Christoforidis et al., 1999b
), presumably until PtdIns(3,4,5)P3 and PtdIns(3,4)P2 are depleted. Other 5-phosphatases, in particular SHIP, that prefers PtdIns(3,4,5)P3 as a substrate, may cooperate with 5-Pase in the first of the two dephosphorylation reactions. This model is consistent with a study by Ivetac et al. (2005)
, published while this manuscript was in revision, that reported the association of 4-Pase with early and recycling endosomes in COS-1 cells.
Interestingly, the 5-Pase was identified in a search for PtdIns(3,4,5)P3-binding proteins (Krugmann et al., 2002
) and the 4-Pase was recovered in a complex with PI3-K (Munday et al., 1999
) and binds PtdIns(3,4)P2 via its C2 domains (Ivetac et al., 2005
). These observations raise the interesting possibility that, at the plasma membrane, Rab5 may regulate the recruitment of various effectors by a combinatorial principle similar to the one operating on early endosomes. Whereas on early endosomes Rab5 regulates the recruitment of FYVE proteins (e.g., EEA1) in combination with PtdIns(3)P, at the plasma membrane it may cooperate with PtdIns(3,4,5)P3 in the recruitment of other effector proteins. Specifically, activated Rab5 would bind and stimulate PI3Kβ activity, thus eliciting in a positive feedback mechanism the production of its “co-receptor” PtdIns(3,4,5)P3. Both Rab5 and PtdIns(3,4,5)P3 would then serve as binding sites to recruit the 5-Pase, resulting in dephosphorylation of PtdIns(3,4,5)P3 to PtdIns(3,4)P2. PtdIns(3,4)P2 would then recruit the 4-Pase, which, activated by Rab5-GTP, would dephosphorylate PtdIns(3,4)P2 to PtdIns(3)P.
The extent to which such enzymatic cascade operates along the pathway probably depends on cell type and growth conditions. Under steady-state, it may contribute only a lesser (<30%) fraction of PtdIns(3)P production in comparison with direct phosphorylation of PtdIns by hVps34. However, it may constitute an important regulatory system ensuring endocytic transport and organelle homeostasis under various PI 3-K–dependent signaling conditions. Rab5 itself is activated both at the plasma membrane and on EEA1-positive early endosomes upon EGF stimulation (Di Fiore and De Camilli, 2001
). The stimulatory activity of Rab5 on PI3Kβ could thus contribute to the generation of 3-PIs at the cell surface in response to various signals, thus inducing morphogenetic changes (Spaargaren and Bos, 1999
; Lanzetti et al., 2004
). The finding that both 5- and 4-Pase are recruited along with Rab5 to the cell cortex upon serum stimulation (this study and Ivetac et al., 2005
) strongly supports the view that the PtdIns(3,4,5)P3 produced is subjected to Rab5-regulated turnover to restrict it to the plasma membrane and to restore the production of PtdIns(3)P before, or at arrival into, the early endosomes that accumulate underneath the ruffling region. This mechanism must operate with high efficiency early in the pathway as both 5- and 4-Pase do not accumulate on early endosomes (), and PtdIns(3,4,5)P3 and PtdIns(3,4)P2 could not be detected on this compartment (). Accordingly, PtdIns(3,4,5)P3 was observed in the phagocytic cup (Marshall et al., 2001
) but not on phagosomes, which were instead enriched in PtdIns(3)P (Vieira et al., 2001
). In addition, Rab5 may regulate the activity of other PI Pases, as we have recently detected the inositol polyphosphate 5-phosphatase OCRL (Attree et al., 1992
; Zhang et al., 1995
) in the eluate from the Rab5 affinity column (unpublished data). By converting PtdIns(4,5)P2 into PtdIns(4)P, OCRL may contribute to the Rab5-dependent regulation of PIs on endosomal receptor trafficking and sorting (Ungewickell et al., 2004
; Choudhury et al., 2005
Although dephosphorylation at the 3′ position of the inositol ring by PI 3-Pases such as PTEN (Leslie and Downes, 2002
) may contribute to termination of PI(3,4,5)P3 signaling, a distinguished feature of the enzymatic cascade described in this study is the generation of other 3-PIs that have signaling functions of their own. Unlike Ivetac et al. (2005)
, we could not consistently observe endosomal abnormalities in HeLa cells lacking 4-Pase or primary cultures of weeble neurons (unpublished data). The expression of inositol 4-phosphatase type II may partially compensate for the lack of 4-Pase (the type I isoform; Majerus et al., 1999
) and the presence of Vps34 ensures the bulk of production of PtdIns(3)P. However, unexpectedly we could detect alterations in receptor-mediated endocytosis. Because inhibition of PI3-K with wortmannin does not dramatically impair transferrin internalization (Martys et al., 1996
; Shpetner et al., 1996
; Spiro et al., 1996
), it is plausible that the accumulation of PtdIns(3,4,5)P3 and PI(3,4)P2 rather than reduced production of PI(3)P on endosomes by 4-Pase RNAi may exert an inhibitory effect on the endocytic process.
Unbalance in PI metabolism and neuronal degeneration in 4-Pase–deficient mice
That the aforementioned turnover of PIs is of high physiological importance is underscored by the phenotypic analysis of weeble mutant mice (Nystuen et al., 2001
). When we inspected the PIs levels in cultured astrocytes of weeble mice, we detected an enhancement of PtdIns(3,4)P2 as well as significant (~30%) reduction in PtdIns(3)P, under both resting and stimulatory conditions. These data are consistent with the measurements on PI synthesis on HeLa membrane fractions in vitro and the view that dephosphorylation of PtdIns(3,4,5)P3 to PtdIns(3)P is impaired in weeble mutants due to a selective block of the 4-Pase reaction.
The precise mechanism leading to early neuronal loss in weeble mutant mice remain to be established. We suggest that impaired PtdIns(3,4)P2 dephosphorylation may cause an imbalance in both signaling and endocytosis due to excess PtdIns(3,4)P2 signaling, lowered PtdIns(3)P production or both. Several scenarios, including the ones listed below, can be hypothesized to explain neuronal death.
First, abnormal PI signaling may affect the balance between factors that promote cell survival and cell death. During postnatal neural development, cell proliferation, apoptosis, and differentiation are regulated by complex and accurately orchestrated signaling pathways, triggered by various neurotrophic factors (Segal and Greenberg, 1996
). PIs play an essential role in these processes (Fruman et al., 1998
) and the unbalance in PI turnover, primarily the accumulation of PtdIns(3,4)P2, may have important disrupting effects of growth factor receptor signaling. For example, neurons that are forced to reenter the cell cycle by an expressed oncogene undergo apoptosis rather than divide (Feddersen et al., 1992
Second, the endocytic alterations due to loss of 4-Pase may produce directly or indirectly an impact on neuronal function. For example, a chronic impairment in glutamate receptor endocytosis may lead to excess excitatory signaling with resulting cell death (Garthwaite and Garthwaite, 1991
; Doughty et al., 2000
Third, as 4-Pase can also act on inositol polyphosphates (Norris et al., 1995
; Majerus et al., 1999
), actions mediated by abnormal metabolic flux through the various inositol polyphosphate metabolites cannot be excluded. Besides Ins(1,4,5)P3, which controls Ca2+
signaling, inositol polyphosphates also have important effects on nuclear function (Steger et al., 2003
; York, 2003
In conclusion, our data underscore the importance of enzymatic networks regulating PI synthesis and turnover to coordinate both signaling and trafficking functions. The Rab5 network of PI kinases and phosphatases appears to be fundamental under stimulatory conditions, particularly during maturation of the nervous system. It will be important to further elucidate how Rab5 and its effectors may actively participate in signal transduction in neurons and other highly differentiated cell types.