Among the phosphatases that hydrolyze inositol polyphosphates, Minpp1 is unique by virtue of three characteristics: an active site histidine, cleavage of the 3-phosphate from multiple inositol polyphosphates, and compartmentalization in the lumen of the ER. In vitro, Minpp1 hydrolyzes Ins(1,3,4,5)P4, InsP5, and InsP6, and kinetic experiments have indicated that InsP5 and InsP6 are the most important substrates for Minpp1. While these two polyphosphates are abundant in the cytosol, localization of Minpp1 to the ER has made it difficult to prove that these substrates gain access to the enzyme in vivo.
One of the major new conclusions to arise from this study is that ER-based Minpp1 does regulate InsP5 and InsP6 levels in vivo. Support for this proposal comes from our observation that in Minpp1−/− MEF the cellular levels of InsP5 and InsP6 were 30 to 45% higher than those of Minpp1+/+ MEF. Reintroduction of Minpp1 into the ER of Minpp1−/− MEF reduced the levels of InsP5 and InsP6 by 35 to 62%, approximately reversing the consequences of the Minpp1 gene deletion. In these add-back experiments, the expression level of exogenous Minpp1 in the ER of Minpp1−/− MEF was considerably greater than the level of endogenous Minpp1 in Minpp1+/+ MEF (data not shown). Moreover, the overexpression of ER-based Minpp1 in NIH 3T3 cells did not reduce levels of InsP5 and InsP6 much below those in wild-type cells. These results strongly suggest the existence of setpoint mechanisms that resist reductions in cellular levels of InsP5 and InsP6 below a critical minimum value. The maintenance of cellular InsP5 and InsP6 may be achieved through a low-affinity transport mechanism that allows access of InsP5 and InsP6 to ER-based Minpp1 only if these compounds rise above a certain concentration; another mechanism could be upregulation of the various kinases that synthesize InsP5 and InsP6 from the precursor Ins(1,4,5)P3.
Further support for the importance of maintaining cellular InsP5
is provided by our experiments with cytosolic expression of Minpp1. It was striking that expression of very low levels of catalytically active Minpp1 in the cytosol substantially lowered InsP5
levels and negatively affected cell growth. Increased levels of InsP5
have been associated with cell cycle progression, although there has been no evidence of a direct link (3
). Thus, our results demonstrate for the first time that maintenance of cellular InsP5
is essential to normal growth of mammalian cells. This conclusion is consistent with recent observations in yeast: depletion of cellular InsP6
slows cell growth and impedes gene expression by decreasing export of mRNA from the nucleus (28
). We can now fully appreciate the importance of the ER membrane barrier in restricting access of Minpp1 to its substrates.
In light of the necessity of ER localization for Minpp1, it becomes all the more intriguing to find that the Minpp1
gene encodes the inositol polyphosphate phosphatase previously localized to the plasma membrane in erythrocytes (11
). Mature mammalian erythrocytes are a specialized cell type in that they have lost their nuclei and ER. They also lack a receptor-activated phospholipase C and do not synthesize inositol phosphates. The situation is different during erythropoiesis. Erythropoietic cells express receptor-regulated phospholipase C activity (24
); consequently, InsP5
seem likely to accumulate from the precursor Ins(1,4,5)P3
. If InsP5
were to be retained by mature erythrocytes, they would bind tightly to hemoglobin and impair the normal regulation of its affinity for oxygen by 2,3-diphosphoglycerate (2
). Therefore, Minpp1 may be responsible for ensuring that InsP5
do not persist in mature erythrocytes. An important clue as to the significance of Minpp1, at least in mature erythrocytes and perhaps during erythropoiesis, is underlined by our demonstration that a substitutive inositol polyphosphate phosphatase is upregulated in the erythrocytes from Minpp1−/−
mice. The identity of this novel enzyme awaits further investigation, but the existence of this phosphatase may explain why hemoglobin content and oxyhemoglobin saturation were unchanged in Minpp1−/−
mice compared to Minpp1+/+
controls (data not shown).
Perhaps this novel inositol polyphosphate phosphatase, and/or other phosphatases, is upregulated in other tissues from Minpp1−/−
mice, although we could not detect any such enzyme activity in liver or brain, at least under the Minpp1 assay conditions. Nevertheless, the presence of alternative mechanisms for regulating levels of InsP5
may be one reason that Minpp1−/−
mice did not exhibit any detectable abnormalities. This lack of a phenotype is otherwise surprising, since Minpp1
is a single-copy gene, the message is widely expressed during mouse development, and the protein possesses a number of unique biochemical features (1
deficiency may also result in subtle phenotypic alterations at the cellular level that we have yet to detect. For example, we considered the possibility that Minpp1 is involved in the regulation of Ca2+
storage and release, and/or the ER stress response, as is the case for other lumenal ER proteins such as calreticulin and BiP/GRP78 (15
). However, no significant differences in agonist-induced cytosolic Ca2+
transients were detected between Minpp1+/+
MEF and pancreatic acinar cells (data not shown). Stress-induced ER-based BiP/GRP78 expression, in response to tunicamycin (10 μg/ml) or thapsigargin (100 nM) treatment for up to 24 h, was indistinguishable between Minpp1+/+
MEF. Also, Minpp1
mRNA expression itself was not regulated by these treatments (data not shown).
In summary, we have provided evidence that Minpp1 participates in the homeostatic regulation of the metabolic pools of InsP5 and InsP6 in vivo. Our data also show that these pools must be maintained for normal cell growth, except in the case of the mature erythrocyte, which employs Minpp1 activity to deplete InsP5 and InsP6. Despite these activities, and a strongly suggested role in chondrocyte differentiation, we have yet to define the upstream and downstream elements in those pathways that employ Minpp1 activity. We are currently performing yeast two-hybrid screens to identify proteins that interact with Minpp1. Upon identification of such proteins, combined with continued growth in the understanding of inositol polyphosphates, our Minpp1−/− mice will provide a valuable genetic background for further study of this novel enzyme.