br Regulation of V ATPase assembly in response to
Regulation of V-ATPase assembly in response to changes in amino Palomid 529 synthesis levels A central regulator of cell growth and metabolism is mTORC1 . mTORC1 integrates signals from nutrient availability and growth factor receptors to control such processes as protein and lipid synthesis and autophagy. Activation of mTORC1 by growth factors requires adequate levels of amino acids, which are sensed at the lysosome. The V-ATPase, in complex with the Ragulator, senses amino acid levels and signals to the Rag GTPases to recruit mTORC1 to the lysosomal membrane, where it is activated by Rheb . Thus, inhibition of the V-ATPase with concanamycin results in inhibition of mTORC1 activity. Recently, our laboratory demonstrated that V-ATPase assembly on lysosomes is controlled by amino acid levels . Thus, amino acid starvation increases both V-ATPase assembly and V-ATPase-dependent lysosomal acidification in a reversible manner . Amino acid-dependent changes in assembly require V-ATPase activity and are blocked by neutralization of the lysosome. Unlike changes in assembly in response to glucose , during dendritic cell maturation  or during influenza infection , , changes in assembly in response to amino acids are not dependent upon either PI-3 kinase or mTORC1 activity . Thus, a novel signaling pathway controls amino acid-dependent changes in V-ATPase assembly (Fig. 2). The fact that amino acid activation of mTORC1 requires V-ATPase activity whereas elevated levels of amino acids actually decrease V-ATPase activity suggests that amino acid-dependent changes in V-ATPase assembly are not involved in changes in mTORC1 activity . In support of this, there is little correlation in the response of V-ATPase assembly and mTORC1 activity to the withdrawal of individual amino acids or to neutralization of lysosomes with chloroquine . We have suggested that the increased V-ATPase-dependent lysosomal acidification that we observe upon amino acid starvation increases lysosomal protein breakdown, thus helping to restore amino acids to normal levels. Interestingly, the V-ATPase-Ragulator complex is also involved in lysosomal recruitment and activation of AMP kinase in response to glucose depletion , although the involvement of changes in V-ATPase assembly in this process is unknown. The V-ATPase is thus an integral part of the nutrient sensing machinery of cells.
Function of V-ATPases in breast cancer cell invasion A variety of studies have suggested that V-ATPases play a role in the survival and invasiveness of tumor cells (Fig. 3) (for reviews see , ). For human breast cancer cells, comparison of the highly invasive MB231 line with the poorly invasive MCF7 line revealed that the highly invasive line expressed much higher levels of V-ATPase at the plasma membrane than the poorly invasive line . Moreover, the in vitro invasiveness of the highly invasive cells but not the poorly invasive cells was inhibited by specific V-ATPase inhibitors (bafilomycin and concanamycin). Targeting of V-ATPases to different cellular membranes is controlled by isoforms of subunit a (in mammals, a1–a4), with a3 and a4 directing V-ATPase complexes to the plasma membrane of osteoclasts and renal intercalated cells, respectively , , , . Our laboratory has shown that MB231 cells express 70-fold higher levels of a3 mRNA and 20-fold higher levels of a4 mRNA relative to MCF7 cells, whereas mRNA levels for a1 and a2 were comparable . Moreover, knockdown of both a3 and a4 using isoform-specific siRNAs inhibited in vitro invasion by MB231 cells , suggesting that expression of a subunit isoforms capable of targeting V-ATPase to the plasma membrane was important in breast cancer cell invasion. In order to compare more closely related human breast cancer cell lines, MCF10a and MCF10CA1a cells were next employed . MCF10a cells are a non-tumorigenic, non-invasive human breast epithelial cell line whereas MCF10CA1a cells were derived by transfection of MCF10a cells with Ras and repeated selection for cells able to metastasize in mice , . Our laboratory showed that the in vitro invasion of the highly metastatic MCF10CA1a cells but not the parental MCF10a cells was inhibited by concanamycin . In addition, MCF10CA1a cells expressed much higher levels of both a3 mRNA and plasma membrane V-ATPases than MCF10a cells. Moreover, siRNA-mediated knock-down of a3 but not other a subunit isoforms significantly inhibited both plasma membrane V-ATPase expression and in vitro invasion of MCF10CA1a cells . Importantly, over-expression of a3 (but not the other a subunit isoforms) in the parental MCF10a cells dramatically increased both invasiveness and the expression of V-ATPases at the plasma membrane . These results suggest that highly invasive tumor cells express high levels of a3 which target V-ATPases to the plasma membrane, where they aid in invasion. How a3 expression is upregulated in tumor cells is not known but is a question of great interest. Transcription factors such as TFEB  have been shown to upregulate expression of V-ATPase subunits, and this is linked to the dependence of many tumors on autophagy as a source of amino acids , but it is not known whether up-regulation occurs in an isoform specific manner. It is also possible that a3 expression is under the control of factors which regulate the expression of genes involved in invasion or migration, but the nature of such factors remains to be elucidated.