Macropinocytosis serves as an internalization pathway for extracellular fluid and its contents and is upregulated in oncogene-expressing cells. Recently, we have revealed a functional role for macropinocytosis in fueling cancer cell growth through the internalization of extracellular albumin, which is degraded into a usable source of intracellular amino acids. Assessing macropinocytosis has been challenging in the past due to the lack of reliable assays capable of quantitatively measuring this uptake mechanism. Here, we describe a protocol for visualizing and quantifying the extent of macropinocytosis in cancer cells both in culture and in vivo. Using this approach, the ‘Macropinocytic Index’ of a particular cancer cell line or subcutaneous tumor can be ascertained within 1–2 days. The protocol can be carried out with multiple samples in parallel and can be easily adapted for a variety of cell types and xenograft/allograft mouse models.
Recent work suggests a link between endocytic trafficking and mTORC1 signaling. This paper demonstrates a specific requirement for the integrity of the late endosomal compartment for amino acid and insulin-stimulated mTORC1 signaling to downstream effectors.
We previously proposed a model of Class IA PI3K regulation in which p85 inhibition of p110␣ requires (i) an inhibitory contact between the p85 nSH2 domain and the p110␣ helical domain, and (ii) a contact between the p85 nSH2 and iSH2 domains that orients the nSH2 so as to inhibit p110␣. We proposed that oncogenic truncations of p85 fail to inhibit p110 due to a loss of the iSH2-nSH2 contact. However, we now find that within the context of a minimal regulatory fragment of p85 (the nSH2-iSH2 fragment, termed p85ni), the nSH2 domain rotates much more freely ( c Ϸ12.7 ns) than it could if it were interacting rigidly with the iSH2 domain. These data are not compatible with our previous model. We therefore tested an alternative model in which oncogenic p85 truncations destabilize an interface between the p110␣ C2 domain (residue N345) and the p85 iSH2 domain (residues D560 and N564). p85ni-D560K/N564K shows reduced inhibition of p110␣, similar to the truncated p85ni-572 STOP . Conversely, wild-type p85ni poorly inhibits p110␣N345K. Strikingly, the p110␣N345K mutant is inhibited to the same extent by the wild-type or truncated p85ni, suggesting that mutation of p110␣-N345 is not additive with the p85ni-572 STOP mutation. Similarly, the D560K/N564K mutation is not additive with the p85ni-572 STOP mutant for downstream signaling or cellular transformation. Thus, our data suggests that mutations at the C2-iSH2 domain contact and truncations of the iSH2 domain, which are found in human tumors, both act by disrupting the C2-iSH2 domain interface.cancer ͉ glioblastoma ͉ phosphoinositide 3-kinase ͉ PIK3CA P I 3-kinases are important cellular regulators of growth, survival, and motility, and deregulation of PI 3-kinase signaling contributes to cancer and other human diseases (1). Class IA PI 3-kinases, which produce PI[3,4,5]P3 in intact cells (2), are obligate heterodimers of a regulatory subunit (p85␣, p85, p55␣, p50␣, or p55␥) and a catalytic subunit (p110␣, p110, or p110␦) (reviewed in ref.3). The regulatory subunits have two major functions: they stabilize the catalytic subunits against thermal denaturation, and they maintain the catalytic subunit in an inhibited, low activity state (4, 5).p85 and p110 are both multidomain proteins that bind to each other and to upstream activators such as Rac and Cdc42, Ras, and tyrosine phosphorylated receptors and adapters (reviewed in ref. 6). p85 contains an SH3 domain, a Rac/Cdc42-binding domain homologous to a GAP domain in the BCR gene product, and two SH2 domains that flank an antiparallel coiled coil domain (the iSH2 domain). While NMR, EPR, and crystal structures have been obtained for the individual domains (7-15), there are currently no structures that define how these domains are arranged in space. The p110␣ catalytic subunit has been better defined, with structures of the N-terminal adapter-binding domain (ABD) or the entire p110␣ bound to the coiled coil (iSH2) domain of p85 (15,16). Like the related Class IB catalytic subunit p110␥ (17), p110␣ contains Ras-binding, C2, ...
The mammalian Class III PI3K (phosphoinositide 3-kinase), hVps34 [mammalian Vps (vacuolar protein sorting) 34 homologue], is an important regulator of vesicular trafficking, autophagy and nutrient sensing. In yeast, Vps34 is associated with a putative serine/threonine protein kinase, Vps15, which is required for Vps34p activity. The mammalian homologue of Vps15p, hVps15 (formerly called p150), also binds to hVps34, but its role in hVps34 signalling has not been evaluated. In the present study we have therefore compared the activity and regulation of hVps34 expressed without or with hVps15. We find that hVps34 has low specific activity when expressed alone; co-expression with hVps15 leads to a marked increase in activity. Notably, beclin-1/UVRAG (UV radiation resistance-associated gene) activation of hVps34 requires co-expression with hVps15; this may be explained by the observation that beclin-1/UVRAG expression increases hVps34/hVps15 binding. Regulation of hVps34 activity by nutrients also requires co-expression with hVps15. Finally, given a recent report that hVps34 activity requires Ca2+/CaM (calmodulin), we considered whether hVps15 might be involved in this regulation. Although hVps34 does bind CaM, we find its activity is not affected by treatment of cells with BAPTA/AM [1,2-bis-(o-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid tetrakis(acetoxymethyl ester)] or W7. Removal of CaM by EDTA or EGTA washes has no effect on hVps34 activity, and hVps34 activity in vitro is unaffected by Ca2+ chelation. The results of the present study show that, in mammalian cells, hVps34 activity is regulated through its interactions with hVps15, but is independent of Ca2+/CaM.
Class IA (p85/p110) phosphoinositide 3-kinases play a major role in regulating cell growth, survival, and motility. Activating mutations in the p110α isoform of the class IA catalytic subunit (PIK3CA) are commonly found in human cancers. These mutations lead to increased proliferation and transformation in cultured cells, but their effects on cell motility and tumor metastasis have not been evaluated. We used lentiviral-mediated gene transfer and knockdown to produce stable MDA-MB-231 cells in which the endogenous human p110α is replaced with either wild-type bovine p110α or the two most common activating p110α mutants, the helical domain mutant E545K and the kinase domain mutant H1047R. The phosphoinositide 3-kinase/Akt pathway was hyperactivated in cells expressing physiologic levels of helical or kinase domain mutants. Cells expressing either mutant showed increased motility in vitro, but only cells expressing the helical domain mutant showed increased directionality in a chemotaxis assay. In severe combined immunodeficient mice, xenograft tumors expressing either mutant showed increased rates of tumor growth compared with tumors expressing wild-type p110α. However, tumors expressing the p110α helical domain mutant showed a marked increase in both tumor cell intravasation into the blood and tumor cell extravasation into the lung after tail vein injection compared with tumors expressing wild-type p110α or the kinase domain mutant. Our observations suggest that, when compared with kinase domain mutations in a genetically identical background, expression of helical domain mutants of p110α produce a more severe metastatic phenotype. [Cancer Res 2009;69(23):8868-76]
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