Phosphatidylinositolphosphate phosphatase activities and cancer

In mammals, the phosphoinositide 3-kinase (PI3K) isoforms responsible for phosphorylating the 3-hydroxyl group of the inositol headgroup of PtdIns, PtdIns(4)P, and PtdIns(4,5)P₂ have been divided into three classes: class I, class II, and class III.

In response to activation of cell surface receptors, class I PI3Ks are responsible for phosphorylating PtdIns(4,5)P₂ to generate PtdIns(3,4,5)P₃ (). There are four class I PI3Ks that exist as heterodimers of regulatory and catalytic subunits. The four distinct catalytic subunits, p110α, -β, -γ, and -δ, are further divided into class IA (p110α, -β, and -δ) and class IB (p110γ). Class IA PI3Ks bind the SH2 domain containing the p85 family of regulatory subunits (p85α, p85β, and p55) that bind to protein tyrosine phosphate residues in activated receptors, thus relieving p85-mediated inhibition of p110 catalytic subunits and facilitating access to PI3K lipid substrates in cell membranes. The single class IB PI3K, p110γ, can bind p84 or p101 regulatory subunits that mediate heterotrimeric G-protein βγ-subunit activation of p110γ (, ).

The class II PI3Ks, PI3K-C2α, PI3K-C2β, and PI3K-C2γ, phosphorylate PtdIns to generate PtdIns(3)P in cells [reviewed in ()]. There is some evidence that class II PI3Ks can be regulated by activated cell surface receptors, but because they do not have regulatory subunits, the mechanism is unclear (). Furthermore PI3K-C2α has been reported to phosphorylate PtdIns(4)P to generate PtdIns(3,4)P₂ ().

The sole class III PI3K in mammals, Vps34, phosphorylates PtdIns in cells to generate PtdIns(3)P [reviewed in ()]. Vps34 is regulated by at least three distinct regulatory complexes ().

Gene amplification of the genes encoding all four p110 catalytic subunits have been reported in several human cancers, including ovarian, prostate, lung, thyroid, cervical, and glioblastoma [reviewed in ()]. Furthermore, the gene encoding p110α, PIK3CA, is frequently mutated in human cancers such as colorectal, breast, lung, and glioblastoma (). The consequence of gene amplification and the mutations in p110 subunits is the increase in the basal activity of PI3Ks.

Somatic mutations have also been identified in the gene encoding the p85α subunit, PIK3R1, in colon, breast, pancreatic, and glioblastoma cancers (). The p85α mutants interact with the p110α, -β, and -δ subunits, but because they have an impaired ability to inhibit the activity of the catalytic subunits, the basal activity of class IA PI3Ks is increased ().

To date, there have been no somatic mutations in the genes encoding the class II and class III PI3Ks in human cancer.

Since the discovery that the serine/threonine kinase, AKT/PKB, is an effector of PI3K in the mid-nineties (, , , , ), the enzyme has been identified as the key transducer of oncogenic signaling in numerous human cancers. Activating mutants of AKT have been identified in human cancers such as breast, colorectal, and ovarian (). The AKT gene family comprises three forms, of which AKT1 and AKT2 are widely expressed, but AKT3 expression appears to be limited to brain tissue. AKT phosphorylates upwards of 200 target proteins that regulate gene expression, protein synthesis, cell cycle progression, cytoskeleton organization, and cell metabolism ().

PtdIns(3,4,5)P₃ and PtdIns(3,4)P₂ generated in response to growth factor stimulation recruit AKT/PKB to the plasma membrane by binding to its N-terminal pleckstrin homology (PH) domain (, ). This interaction has been shown to activate AKT in vitro (, , ). However, other studies have favored a model in which PtdIns(3,4,5)P₃ and PtdIns(3,4)P₂ binding does not activate AKT (), but instead recruits the enzyme to the membrane, altering its conformation and allowing the kinase to be fully activated by subsequent phosphorylation at Thr308 and Ser473.

AKT is phosphorylated at Thr308 in the activation loop by a PH domain containing 3-phosphoinositide-dependent kinase 1 (PDK1) (, , , ). PDK1 only phosphorylates AKT in the presence of PtdIns(3,4,5)P₃ or PtdIns(3,4)P₂ (, , , ). The C-terminal PH domain of PDK1 binds to PtdIns(3,4,5)P₃ and PtdIns(3,4)P₂, with weaker binding to PtdIns(4,5)P₂, resulting in PDK1 binding to the plasma membrane (, ). AKT does not bind PtdIns(4,5)P₂ (). AKT is further activated by phosphorylation at Ser473 in the hydrophobic motif by mTORC2 (). However, little is known about the upstream activators of mTORC2 in response to growth factors. Interestingly, one report has demonstrated that PtdIns(3,4,5)P₃ can directly activate mTORC2 activity in vitro ().

There is evidence that PDK1 can be recruited to membranes independently of PtdIns(3,4,5)P₃ or PtdIns(3,4)P₂ through its binding to the activated insulin receptor via the adaptor protein, Grb14 (). Furthermore, the PH domain of PDK1 exhibits a greater binding affinity for PtdIns(3,4,5)P₃, PtdIns(3,4)P₂, and PtdIns(4,5)P₂ than the binding affinity of the PH domain of AKT for PtdIns(3,4,5)P₃ (, ). Indeed, the binding affinity of PDK1 for PtdIns(4,5)P₂ is comparable to the affinity of AKT toward PtdIns(3,4,5)P₃ (). Therefore, AKT membrane localization and activation is potentially more dependent on higher PtdIns(3,4,5)P₃ and PtdIns(3,4)P₂ levels than the levels necessary for PDK1 localization. In this model, PtdIns(3,4,5)P₃ and PtdIns(3,4)P₂ are considered to have redundant abilities to activate AKT and PDK1; however, one study has suggested that both PtdIns(3,4,5)P₃ and PtdIns(3,4)P₂ are required for full activation of AKT ().

Not all PI3K signals in cancer are transduced by AKT phosphorylation. The levels of phosphorylated AKT in PIK3CA mutant cell lines and human breast tumors are low and anchorage-dependent growth is less dependent on AKT (). However, PDK1 is highly expressed in PIK3CA mutant breast tissue, and PDK1 expression is required for anchorage-independent growth in PIK3CA mutant cell lines (). PDK1 is not solely an AKT kinase; it phosphorylates the activation segment of at least 23 AGC kinases [reviewed in ()]. One of these kinases, the serum and glucocorticoid-regulated kinase 3 (SGK3) is phosphorylated by PDK1 at the activation loop () and mTORC2 at the hydrophobic motif (). SGK3 is amplified and hyperactivated in PIK3CA-mutant breast cancer (), and is required for AKT-independent viability (), anchorage-independent growth, and spheroid growth ().

Over the last 20 years, there has been a tremendous amount of research that has demonstrated that PI3K/AKT signaling is constitutively activated in a large number of human cancers as a result of gene mutation or amplification of PI3K and AKT. However, the functional loss of the phosphoinositide phosphatases has also been reported to drive the progression of human cancer. There are phosphatases which remove the phosphate group from each of the 3-, 4-, and 5-positions of PtdIns(3,4,5)P₃ and related phosphoinositides. Considering the key role the PI3K/AKT pathway plays in malignancy, changes in the activities of these enzymes will have distinct effects upon the onset and progression of cancer. Thus, this review will highlight the phosphoinositide phosphatase activities expressed in humans and consider their involvement in cancer, recognizing that most data relate to the 3- and 5-phosphatases.

Mammalian phosphoinositide 3-phosphatases can be divided into two major groups: one group comprised of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and the transmembrane phosphatase with tensin homology (TPTE) and PTEN homologous inositol lipid phosphatase (TPIP) proteins, and the other group made up of myotubularin (MTM) and MTM-related (MTMR) proteins.

PTEN, also known as mutated in multiple advanced cancers (MMAC), was first identified as a candidate tumor suppressor gene on chromosome 10q23 in 1997 (, ). PTEN is the best known and most described of phosphoinositide phosphatases that play a role in human malignancy. Somatic homozygous mutations and deletions of PTEN have been detected in glioblastoma, prostate, kidney, melanoma, lung, endometrial, bladder, and breast cancer cell lines and primary tumors (, , , , , ). That same year, it was reported that germline mutations of PTEN were associated with Cowden's disease, an autosomal dominant cancer predisposition syndrome that has an associated increased risk of cancer (, ). Homozygous PTEN knockout mice are embryonic lethal, but PTEN/− mice are viable (, , ). Nevertheless, consistent with the tumor suppressor function of PTEN and the situation observed in Cowden's disease patients, heterozygous PTEN/− mice exhibited a higher frequency of spontaneous tumor formation compared with wild-type mice (, , ).

Due to its similarity to a subfamily of protein tyrosine phosphatases, the dual specificity phosphatases, in particular the VH1-like family (), PTEN was initially predicted to be a dual specificity protein phosphatase (, , ). Subsequent work demonstrated that recombinant PTEN exhibited tyrosine phosphatase (, , ) and serine/threonine phosphatase activity in vitro (). Importantly, Myers et al. () demonstrated that mutation of the essential cysteine from the invariant signature motif HCxxGxxR found in all the members of the protein tyrosine phosphatase superfamily, PTEN C124S, resulted in loss of tyrosine phosphatase activity in vitro. Consistent with the in vitro results, overexpression of PTEN in NIH-3T3 cells reduced tyrosine phosphorylation of focal adhesion kinase in vitro and in vivo (). Furthermore, when the phosphatase inactive PTEN C124S mutant was overexpressed in NIH-3T3 and glioblastoma U-89 cell lines, focal adhesion kinase tyrosine dephosphorylation, cell spreading, invasion, and growth were attenuated (, ).

Maehama and Dixon () suggested that PTEN could dephosphosphorylate negatively charged substrates other than tyrosine and/or serine/threonine phosphoproteins. They based this on the observation that the in vitro catalytic rate activity of PTEN toward protein and peptide substrates was low (, , ), but was far greater toward highly negatively charged and multi-phosphorylated tyrosine peptide substrates such as polyGlu4Tyr1 (). When PTEN was overexpressed in HEK293 cells, the levels of insulin-stimulated PtdIns(3,4,5)P₃ were found to be significantly reduced, whereas overexpression of the catalytically inactive PTEN C124S mutant resulted in an increase in cellular PtdIns(3,4,5)P₃ in the absence of insulin stimulation (). Furthermore, recombinant PTEN dephosphorylated PtdIns(3,4,5)P₃ to PtdIns(4,5)P₂ and Ins(1,3,4,5)P₄ to Ins(1,4,5)P₃ in vitro (), confirming that PTEN was indeed a lipid phosphatase with activity toward phosphates at the 3-position of the inositol ring.

Myers et al. () confirmed that recombinant PTEN catalyzed the dephosphorylation of PtdIns(3,4,5)P₃ to PtdIns(4,5)P₂ and Ins(1,3,4,5)P₄ to Ins(1,4,5)P₃ in vitro. However, they also demonstrated that in addition to PtdIns(3,4,5)P₃, PTEN was able to dephosphorylate PtdIns(3)P and PtdIns(3,5)P₂ at the 3-position in vitro (), in the order of preference PtdIns(3,4,5)P₃ = PtdIns(3,4)P₂ > PtdIns(3)P > Ins(1,3,4,5)P₄ ().

Interestingly, it was demonstrated that the dephosphorylation activity of the PTEN mutant found in Cowden's disease, PTEN G129E (), against phosphotyrosine peptides is unaltered (), but the activity is significantly reduced against PtdIns(3,4,5)P₃ ().

This result strongly suggested that it is the lipid, rather than the protein phosphatase activity of PTEN that is required for its tumor suppressor function, and that loss of PtdIns(3,4,5)P₃-phosphatase activity results in Cowden's disease (). Furthermore, in immortalized mouse embryonic fibroblasts isolated from PTEN −/− mice, the levels of PtdIns(3,4,5)P₃ are elevated and AKT is hyperactivated (), and in heterozygous PTEN/− mice, the elevated frequency of spontaneous tumor formation is associated with an increased activity of AKT (). These and subsequent studies have shown that the mechanism underlying the tumor suppressor function of PTEN is the regulation of the cellular pool of PtdIns(3,4,5)P₃ levels that controls AKT-mediated signaling [reviewed in ()].

In the Cowden PTEN G129E mutant, the protein phosphatase activity is attenuated, but the PtdIns(3,4,5)P₃ phosphatase activity is intact. Thus, the significance of the PTEN protein phosphatase activity for PTEN tumor suppressor function is not clear.

Nevertheless, it has been suggested that this protein phosphatase activity may be relevant to various physiological functions of PTEN, such as regulating cell migration and invasion. In contrast, PTEN Y138C, found in small cell lung carcinoma, dephosphorylates PtdIns(3,4,5)P₃, but has no protein phosphatase activity (). When overexpressed in a glioblastoma cell line, PtdIns(3,4,5)P₃ levels and AKT phosphorylation are reduced, but cellular invasion into matrigel is not suppressed (), arguing against a role for the protein dephosphorylating activity of PTEN in this function. PTEN Y138C displays increased phosphorylation at Thr366. It has therefore been hypothesized to alter PTEN binding to other proteins, and consequently to altering the localization of PtdIns(3,4,5)P₃ dephosphorylation (), thereby affecting the physiological consequence of a local change in PtdIns(3,4,5)P₃ concentration. The PTEN G129E mutant also displays increased phosphorylation at Thr366. This has been proposed to provide a mechanism whereby the PTEN protein phosphatase activity is required to auto-dephosphorylate PTEN and thereby control lipid phosphatase activity in vivo (). Of potential importance, however, remains the demonstration that PTEN is a protein tyrosine phosphatase for IRS1, both in vitro and in vivo ().

In addition to post-translational modification, PTEN activity can be regulated by intracellular localization and recruitment. In particular, PTEN has been demonstrated in both nuclear and mitochondrial compartments, in addition to being found in the cytoplasm and being translocated to the plasma membrane. The nuclear localization of PTEN appears to be regulated by sumoylation of PTEN (). The function of nuclear PTEN activity is not fully clear, there are reports of nuclear PI3K activity (), and phosphorylated nuclear AKT has been detected in thyroid adenomas and carcinomas with an increase in phosphorylation detectable in PTEN-null tumors (). However, He et al. () demonstrated that PTEN suppresses translocation of phosphorylated AKT, suggesting that it is the non-nuclear rather than the nuclear PTEN that is critical in cancer. Nevertheless, there is clearly a signaling role for nuclear PtdIns(3,4,5)P₃. Blind et al. () have demonstrated that PtdIns(3,4,5)P₃ binds to and stabilizes the nuclear receptor SF-1 structure, explaining the PI3K-dependent increase of SF-1 activity (). This group has, however, questioned the importance of PI3K in this process by demonstrating that PtdIns(4,5)P₂-associated SF-1 can be modified by IMPK, as well as PTEN. A further role for nuclear PTEN is suggested by its interaction with histone H1 via its C2 domain, thereby regulating chromatin condensation and gene expression (). The PTEN-histone H1 interaction is not dependent on PTEN phosphatase activity (), and is thus mediated by protein-protein interaction; the regulation of this process and its regulatory significance in cancer tissues remains unclear.

Mitochondrial PTEN has also been reported, though it has been shown that this is a 70 kDa PTENα which has a 173 amino acid elongated N-terminal region generated through an alternate translation initiation site (). This form of PTEN is detectable in both the cytoplasm and mitochondria, and in the latter localization it can activate cytochrome oxidase and thus mitochondrial oxidative metabolism. It appears that PTENα physically associates with cytochrome oxidase and that mutagenesis demonstrates that the phosphatase activity is necessary for activation of the oxidase. However, while it has been suggested that cytochrome oxidase activity can be regulated by phosphorylation, with the hypo-phosphorylated form showing greater activity (), the mutation in PTENα, C297S, that reduces oxidase activity, is equivalent to C124S in PTEN, and thus regulates lipid rather than protein phosphatase activity. The presence of PtdIns(4,5)P₂ in mitochondria has been reported (); however, there is a paucity of evidence for a mitochondrial PI3K, which nevertheless may exist and there could therefore be an as yet unrecognized PtdIns(3,4,5)P₃ signaling pathway in mitochondria which could be important in malignancy where energy metabolism is increased.

Hopkins et al. () have also reported a longer form of PTEN, which they named PTEN-long. The additional 174 N-terminal amino acids of PTEN-long share homology to the poly-basic residues of the cell-penetrating element of the HIV transactivator of transcription (TAT) protein (, ). Consequently, PTEN-long is a membrane-permeable enzyme that is secreted and can enter other cells to antagonize PI3K-AKT signaling (). PTEN-long is most likely identical to PTENα however, as discussed by Liang et al. (), due to the absence of peptide sequence data for PTEN-long, this remains to be definitely proven. Furthermore, Liang et al. () have proposed, based on sequence analysis and the CUG initiation mechanism, that multiple longer forms of PTEN are likely to be expressed. PTEN-long is not the only PtdIns(3,4,5)P₃ phosphatase that has been reported to be secreted from cells, PTEN has also been reported to be secreted from cells inside microvesicles, called exosomes, allowing PTEN to enter and antagonize PI3K-AKT signaling in other cells (). However, it remains unclear what the physiological function of PTEN and PTEN-long transfer between cells fulfils.

Phosphorylation of residues S380, T382, T383, and S385 in the C terminus of PTEN maintains the phosphatase as a monomer (, ). However, when these residues are not phosphorylated, PTEN exists as a dimer that has greater PtdIns(3,4,5)P₃ phosphatase activity than the monomer (). Furthermore, catalytically inactive mutants of PTEN heterodimerize with wild-type PTEN, resulting in the inhibition of the PtdIns(3,4,5)P₃ phosphatase activity in a dominant-negative manner (). This, in turn, results in hyper-activation of PI3K-AKT signaling and increased tumor formation in mice (). The implications for the clinical outcome of patients that express both wild-type and mutant PTEN proteins are significant based on the dominant-negative effect of the mutant protein. For example, patients with one mutant gene expressing an inactive mutant of PTEN could conceivably have a significant reduction in the PtdIns(3,4,5)P₃ phosphatase activity of the remaining wild-type PTEN, whereas patients expressing PTEN mutants that result in reduced protein levels, would not ().

The dominant-negative effects of cancer-associated PTEN mutants are most likely not just confined to the same cell. The discovery that PTEN is sorted into exosomes, and that PTEN-long is secreted PtdIns(3,4,5)P₃ phosphatases that enter other cells to regulate PI3K-AKT signaling, raises the intriguing possibility that catalytically inactive mutants of PTEN could also be transferred from cancer cells to stromal cells. This cell-to-cell communication of cancer-associated PTEN mutants, and the dominant-negative manner in which they can inhibit the catalytic activity of wild-type PTEN could further accentuate tumor progression by hyperactivating PI3K-AKT signaling in the surrounding stromal cells, thus facilitating the formation of a tumor microenvironment that is required to support tumor growth.

Human cells express a single TPTE (). However, the human enzyme lacks PtdIns(3)P phosphatase activity (). In contrast, mouse TPTE has been demonstrated to exhibit phosphatase activity in vitro toward PtdIns(3)P, PtdIns(3,4)P₂, PtdIns(3,5)P₂, and PtdIns(3,4,5)P₃ (). TPIP exists as four splice variants (TPIPα, -β, -γ, and -C2) (, , ). TPIPα, but not TPIPβ, dephosphorylates PtdIns(3)P, PtdIns(3,4)P₂, PtdIns(3,5)P₂, and PtdIns(3,4,5)P₃ in vitro (), and in contrast to PTEN, TPIPα does not dephosphorylate Ins(1,3,4,5)P₄ in vitro (). To date, there are no reports about whether either TPIPγ or TPIPC2 have phosphatase activity. Although both TPTE and TPIP phosphatases have the capacity to dephosphorylate PtdIns(3,4,5)P₃, there are no reports, to date, that the genes encoding either activity are mutated or overexpressed in a human cancer; thus, it is likely that these enzymes play a limited role in regulating PtdIns(3,4,5)P₃ levels physiologically and their normal physiological role(s) remains open to question.

The PTEN-like phosphatase (PTPMT1) was first identified from database searches using the PTEN active site as a query (). The recombinant protein exhibited phosphatase activity against PtdIns(5)P, but no other phosphoinositide (). However, other reports have demonstrated that PTPMT1 can dephosphorylate PtdIns(3,5)P₂, PtdIns(3,4)P₂, and PtdIns(5)P in vitro (). Furthermore, PtdIns(5)P is not dephosphorylated in vivo by this enzyme (), but rather phosphatidylglycerolphosphate is the physiological substrate (, ); this is a lipid structurally very similar to PtdIns(5)P (). This finding questions the importance of PTPMT1 in regulating the levels of cellular phosphoinositides.

However, Shen et al. () have reported that PtdIns(3,5)P₂ can be dephosphorylated in vivo by the phosphatase. Knockdown of PTPMT1 in cancer cells causes apoptotic death. This is probably brought about through the dual specificity phosphatase being mitochondrially located and its inhibition affects cardiolipin levels leading to modulation of energy metabolism. Nevertheless, this result could suggest that inhibition of this phosphatase could be used in the treatment of cancer (); however, there are no reports that PTPMT1 is mutated in cancer tissues.

MTM1 was first identified as the gene mutated in X-linked recessive myotubular myopathy (). MTM1 was first predicted to be a dual-specificity tyrosine phosphatase (), and initial in vitro assays with recombinant protein reported that MTM1 exhibited both phosphotyrosine (, ) and phosphoserine () dephosphorylating activity. However, Taylor, Maehama, and Dixon () noted that the catalytic activity of MTM1 toward artificial phosphotyrosine substrates was poor, and reported that the active site of MTM1 shares some similarity to the active site region of suppressor of actin (Sac)1, a phosphoinositide phosphatase. Based on these observations, the authors tested the capability of recombinant MTM1 in dephosphorylating phosphoinositides and inositol phosphates, and reported that the enzyme exhibited greatest activity toward PtdIns(3)P and Ins(1,3)P₂ (). However, the soluble inositol phosphate substrate is dephosphorylated with a 10- to 20-fold reduced rate compared with the lipid substrate, which is thus likely to be the authentic substrate in cells (, ). Blondeau et al. () also demonstrated that MTM1 dephosphorylated PtdIns(3)P in vitro and, based on cell expression studies using Schizosaccharomyces pombe, reported that MTM1 dephosphorylated PtdIns(3,5)P₂ in vivo. Subsequent studies demonstrated that MTM1 could dephosphorylate PtdIns(3)P (, , , ) and PtdIns(3,5)P₂ (, ), both in vitro and in vivo.

The MTMR proteins are close homologs of MTM. MTMR1 can dephosphorylate both PtdIns(3)P (, ) and PtdIns(3,5)P₂ () in in vitro assays. There are also reports that MTMR1 dephosphorylates both lipids in vivo. MTMR2 is also able to dephosphorylate PtdIns(3)P in vitro (, , , ) and in vivo () and has also been shown to dephosphorylate PtdIns(3,5)P₂ in vitro (, ). In addition, MTMR2 can dephosphorylate Ins(1,3)P₂, but with a 10-fold lower activity than when dephosphorylating the lipid substrates, making its physiological relevance questionable (). MTMR3 dephosphorylates PtdIns(3)P in vitro (, , , ) and in vivo (), and PtdIns(3,5)P₂ in vitro and in vivo (, ). MTMR4 has been reported to dephosphorylate PtdIns(3)P in vitro () and in vivo (). There are, however, no reports that PtdIns(3,5)P₂ can be a substrate. MTMR6 dephosphorylates PtdIns(3)P in vitro (, , ), and PtdIns(3,5)P₂ both in vitro (, ) and in vivo (). Nevertheless, there are no reports that MTMR6 can dephosphorylate PtdIns(3)P in vivo. The catalytically inactive MTMR9 is able, however, to regulate the activity of MTMR6 and, as such, determines the substrate specificity in vivo (). MTMR7 dephosphorylates PtdIns(3)P in vitro (), and MTMR9 has been shown to increase the in vitro activity (). MTMR8 dephosphorylates PtdIns(3)P and Ptd(3,5)P₂ in vitro, but has only been shown to be active against PtdIns(3)P in vivo (). MTMR9 regulates activity of MTMR8 in vitro and in vivo, and determines substrate specificity in vivo (); it is thus a regulatory component rather than an activity, per se. MTMR14 dephosphorylates PtdIns(3)P and PtdIns(3,5)P₂ in vitro, but only PtdIns(3)P in vivo (). These PtdIns(3)P and PtdIns(3,5)P₂ substrate specificities, with no reported activities against PtdIns(3,4,5)P₃, PtdIns(3,4)P₂, or PtdIns(4,5)P₂, make the importance of these phosphoinositide phosphatases in the regulation of cell proliferation and malignancy questionable, and indeed no tumors have been reported to exhibit mutation or altered expression of the MTMRs.

Dephosphorylation of phosphoinositides at the 4-position is relatively infrequent. Many of the enzymes that catalyze this reaction were identified primarily as inositol polyphosphate phosphatases (INPPs) and, subsequently, as lipid phosphatases.

INPP4A dephosphorylates PtdIns(3,4)P₂ at the 4-position in vitro () and in vivo (), and, to lesser extent, demonstrates activity against Ins(3,4)P₂ and Ins(1,3,4)P₃ in vitro (). The C2 domain of INNP4A is able to bind PtdIns(3,4)P₂, PtdIns(4)P, and phosphatidylserine (). Furthermore, recombinant INPP4A lacking the C2 domain exhibits significantly reduced activity toward PtdIns(3,4)P₂ in vitro, suggesting that this domain regulates substrate binding ().

INPP4A overexpression in cells has been shown to reduce PtdIns(3,4)P₂ (), suggesting that INPP4A might function to suppress AKT activity. Subsequent work using INPP4A −/− mouse embryonic fibroblasts (MEFs) demonstrated that the level of phosphorylation of AKT at both Thr308 and Ser473 in response to EGF was elevated compared with INPP4A +/+ MEFs ().

Furthermore, loss of INPP4A resulted in increased cell growth, decreased apoptosis, and increased anchorage-independent growth, and formed tumors in nude mice, in keeping with activation of the AKT signaling cascade (). INPP4A is thus similar in effect to PTEN in regulating the AKT pathway and cell growth and malignancy.

INPP4B also dephosphorylates PtdIns(3,4)P₂ at the 4-position, and to lesser extent both Ins(3,4)P₂ and Ins(1,3,4)P₃ in vitro () and PtdIns(3,4)P₂ in vivo (). The C2 domain of INNP4B selectively binds PtdIns(3,4,5)P₃ and phosphatidic acid, suggesting that these lipids may play a role in regulating the localization and/or activity of INPP4B in vivo (). However, this hypothesis remains to be tested. More recently, INNP4B has been demonstrated to dephosphorylate PtdIns(3,4,5)P₃ and PtdIns(3,4)P₂ in vitro, showing a greater preference for PtdIns(3,4)P₂ (). Furthermore, from cellular expression experiments, INPP4B has been shown to selectively dephosphorylate PtdIns(3,4)P₂ in vivo (). The knockdown of INPP4B in a human mammary epithelial cell line resulted in anchorage-independent growth, increased cell migration, and elevated AKT phosphorylation in response to insulin (). Furthermore, increased INPP4B expression resulted in reduced tumor growth in a xenograft mouse model; whereas, loss of INPP4B expression has been found to correlate with poor patient survival in both breast and ovarian cancer (). The phosphatidic acid binding of INPP4B, if inhibitory or localizing, could further suggest a cooperative effect with increased phospholipase D activity, which has also been suggested to play a role in breast cancer, particularly metastasis ().

Fedele et al. () reported that decreased INPP4B expression in human breast cancer cell lines elevates AKT signaling and results in increased tumor formation. In keeping with this, INPP4B expression is frequently lost in human breast cancer (). A reduction in expression of INPP4B in prostate cancer cell lines increases AKT phosphorylation and stimulates cell proliferation (), whereas overexpression of INPP4B inhibits prostate cancer cell invasion (). INPP4B expression is reduced in prostate cancer, and patients with lower expression of INPP4B show a decrease in recurrence-free survival (, ). These results demonstrate that INPP4B, and probably INPP4A, is a tumor suppressor that inhibits PI3K/AKT signaling.

TMEM55A (also known as type II PtdIns 4,5-bisphosphate 4-phosphatase) and TMEM55B (also known as type I PtdIns 4,5-bisphosphate 4-phosphatase) both contain two putative transmembrane domains at the C terminus, and they dephosphorylate PtdIns(4,5)P₂ at the 4-position in vitro (). TMEM55B has been further reported to dephosphorylate PtdIns(4,5)P₂ in vivo (, ). In keeping with TMEM55 being unlikely to modulate the AKT pathway, there are no reports of TMEM55 involvement in cancer.

The phosphoinositide 5-phosphatases are grouped into four types (I–IV).

The 40 kDa type I inositol polyphosphate 5-phosphatase, also known as INPP5A, dephosphorylates Ins(1,4,5)P₃ and Ins(1,3,4,5)P₄ at the 5-position in vitro, but has no activity toward phosphoinositides (, , ). There have been a number of studies that suggested a link between INPP5A activity and cancer. However, siRNA knockdown of INPP5A resulted in increased cellular levels of Ins(1,4,5)P₃ and Ins(1,3,4,5)P₄, leading to cell transformation and tumor formation in nude mice (). In addition, INPP5A expression is reduced in human cutaneous squamous cell carcinoma tumors (). Together these observations suggest that, if important, INPP5A will play a tumor suppressing rather than malignancy promoting role.

The type II 5-phosphatases include INPP5B, oculocerebrorenal syndrome (OCRL) (INPP5F), synaptojanin 1 (SYNJ1/INPP5G), synaptojanin 2 (SYNJ2/INPP5H), INPP5J, and INPP5K [skeletal and muscle enriched INPP (SKIP)].

INPP5B, also known as 75 kDa type II inositol polyphosphate 5-phosphatase, dephosphorylates PtdIns(3,4,5)P₃ (, ), PtdIns(4,5)P₂ (, , ), Ins(1,4,5)P₃ (, , ), and Ins(1,3,4,5)P₄ () in vitro. Furthermore, INPP5B has been reported to dephosphorylate PtdIns(4,5)P₂ in vivo (). INPP5B does not dephosphorylate PtdIns(3,5)P₂ (). INPP5B has a similar domain organization to OCRL/INPP5F. However, INPP5B does not contain clathrin binding sites, and it has a C-terminal CAAX prenylation sequence (). To date no human disease or cancer has been associated with INPP5B.

Lowe syndrome, also known as OCRL, is a rare human X-linked developmental disorder that affects brain, kidney, and eye function [reviewed in ()]. Mutations in the OCRL gene were demonstrated to be responsible for Lowe syndrome, and the OCRL protein was found to share similarity with INPP5B ().

Patients diagnosed with a related X-linked disorder, called Dent-2 disease, also carry mutations in OCRL ().

OCRL dephosphorylates Ins(1,4,5)P₃ and Ins(1,3,4,5)P₄ at the 5-position, and PtdIns(4,5)P₂ in vitro, with a greater preference for the lipid substrate (). Subsequent studies demonstrated that OCRL can also dephosphorylate PtdIns(3,4,5)P₃ in vitro (). However, PtdIns(3,5)P₂ is a very poor substrate for INPP5B in vitro (). Studies using cell lines isolated from patients diagnosed with Lowe syndrome have shown that OCRL dephosphorylates PtdIns(4,5)P₂ in vivo (). Despite its similarity in elevating phosphoinositide signaling, there is surprisingly no association between OCRL and cancer.