Ceramide-activated protein phosphatase
Ceramide-activated protein phosphatases (CAPPs) are a subset of intracellular serine/threonine phosphatases which are activated by ceramide lipids. CAPPs have been identified within the protein phosphatase 2A family (PP2A) and the protein phosphatase 1 (PP1) family. Each CAPP consists of a catalytic subunit which confers phosphatase activity and a regulatory subunit which confers substrate specificity. CAPP involvement has been implicated in apoptotic and proliferative pathways related to cancer and other cellular pathways related to Alzheimer’s disease.
Structure
Ceramide-activated PP2A
As a member of the PPA2 family, CAPPs can consist of one to three PPA2 subunits (Janssens & Goris, 2001). The core enzyme consists of a conserved 34 kilodalton (kDa) catalytic subunit, C, and a conserved 65 kDa scaffold subunit, A, tightly bound to one another (Janssens & Goris, 2001). The scaffold subunit contains 15 tandem HEAT repeats which arrange to form a horseshoe-like structure that confers remarkable conformational flexibility (Groves, Hanlon, Turowski, Hemmings & Barford, 1999). Each repeat contains a pair of highly conserved antiparallel alpha helices which form a contiguous ridge (Groves et al., 1999). The catalytic subunit recognizes and associates with the scaffold subunit through this ridge (Groves et al., 1999). Two other regulatory subunit families, the 55 kDa B family and the 61 kDa B' family, can both bind the core enzyme in a mutually exclusive manner (Janssens & Goris, 2001). The B’ subunit is similar in structure to the scaffold subunit and makes extensive interactions with the scaffold subunit, through a convex surface with eight HEAT repeats, and the catalytic subunit (Janssens & Goris, 2001). A concave, acidic region of the B’ subunit is tilted towards the active site of the catalytic subunit in the holoenzyme (Janssens & Goris, 2001). The B subunit contains seven WD40 repeats, a β-hairpin handle and other secondary structures that form a β-propeller (Janssens & Goris, 2001). The β-propeller and β-hairpin handle interact with HEAT repeats three to seven and one to two on the scaffold subunit, respectively (Janssens & Goris, 2001). The B subunit has little interaction with the catalytic subunit but also contains an acidic substrate binding site positioned near the active site of the catalytic subunit (Janssens & Goris, 2001). The B subunits share no sequence identity with B’ subunits (Janssens & Goris, 2001). CAPPs can be present as the core dimeric enzyme of AC or a trimeric holoenzyme of ABC or AB’C (Janssens & Goris, 2001). Ceramide has also been shown to activate the C subunit alone (Janssens & Goris, 2001). There are two isoforms, α and β, of the C and A subunits (Janssens & Goris, 2001). There are four isoforms, α, β γ and δ, of the B subunit and five isoforms α, β, γ, δ, and ɛ of the B’ subunit (Janssens & Goris, 2001). The combinations of these isoforms give a possibility of 40 distinct CAPPs.
Ceramide-activated PP1
Long-chain ceramides have also been shown to activate members of the PP1 family (Chalfant et al., 1999). The CAPPs in this family are composed of a catalytic subunit that can associate with one of over a dozen regulatory subunits (Egloff et al., 1997). The regulatory subunits interact with the catalytic subunit through a conserved RVXF (arginine/lysine-valine/isoleucine-X-phenylalanine) motif (Egloff et al., 1997). When this binding site is deleted from regulatory proteins, they lose the ability to associate with the catalytic subunit (Egloff et al., 1997). There is only one recognition site on the catalytic subunit, making the association of a regulatory subunit mutually exclusive (Egloff et al., 1997). The catalytic subunit of PP1 CAPPs is a single-domain protein consisting of a central β-sandwich, of two mixed β-sheets, with seven α-helices surrounding the sandwich on one side and a sub-domain of consisting of three α-helices and a β-sheet on the other side (Egloff et al., 1997). Three loops that connect β-sheets with α-helices in the top β-sandwich strand form a β α β α β motif that interacts with loops form the opposite β-sandwich sheet to provide the catalytic residues (Egloff et al., 1997).
Activators and inhibitors
In general, PP2A expression is controlled by an auto-regulatory translational mechanism and with developmental regulation of PP2A subunits. However, there are two PP2A inhibitors and only 3 identified PP2A activators that all seem to act on the PP2A enzyme itself. PP2A activators are Theophylline, Sodium selenite and methylation. Mechanisms for all three modes of activation are unknown and more research is needed to explore new activators and to identify new PP2A activators. I1PP2A and I2PP2A are inhibitors of PP2A. I1 PP2A and I2 PP2A inhibit all possible forms of holoenzyme PP2A by directly binding to its catalytic sub unit. Both inhibitors are potent and non competitive(Li, Makkinje, & Damuni, 1996). Both may inhibit using their C terminal tails. It is also suggested that both inhibitors are conserved between mice and humans and both inhibitors are about the same weight (Janssens & Goris, 2001).
I2 PP2A
I2 PP2A does not inhibit PP2A in vitro (instead it produces overexpression) but has been proven to inhibit PP2A in vivo (Janssens & Goris, 2001) . The increased Mg2+ and increased I1PP2A levels in vivo could be needed elements for I2PP2A to act as a PP2A inhibitor. I2PP2A mainly acts as an inhibitor by binding to PP2A's catalytic subunit and changing the subunits geometry (making it non functional). However, I2 PP2A has also been identified as a truncated SET protein. It is then also possible that SET-CAN binding could involve I2 PP2A and thus can inhibit normal PP2A regulation. There are also studies that show leukemic fusion proteins associate with SET and co-immunopreciptate with PP2A. This finding may suggest that I2 PP2A and its inhibitory role of PP2A may play a role in lower cell growth in leukemia (Zhu et al., 2006).
I1 PP2A
I1 PP2A is a PP2A inhibitor in both in vitro and in vivo and is identified as a PHAP-1 protein. I1PP2A has a highly acidic C-terminal tail and the N-terminus is leusine/isoleucine rich (Li et al., 1996). It is also thought that I1 PP2A plays a role in mouse cerebellum development. I1 PP2A co-localizes with the alpha 3A integrin subunit in PC12, in the neurites and Purkinje cells. When I1 PP2A is over expressed, researchers observe a reduction in neurite length. (Mutz et al., 2006) I1 PP2A also seems to be expressed throughout the brain and in brain stem cells. However, expression is higher in cerebellum (Kovacech et al., 2007) I1 PP2A may also indirectly regulate tau and could contribute to processes that cause related tauopathies. Additionally, increased levels of I1 PP2A are associated and play a role in Alzheimer’s disease (Wang, Blanchard, Tung, Grundke Iqbal, & Iqbal, 2015).
Sodium selenate
How sodium selenate specifically activates PP2A has not been well studied. However, the consequences of increased PP2A because of sodium selenate have been studied. Sodium selenate may be a possible treatment for mild brain injury. Sodium selenate increases PP2A activity and increases tau phosphorylation(Tan et al., 2016). . In addition, a sodium selenate induced increase in PP2A also inhibits the PI3K/AKT pathway. Because of this inhibition, sodium selenate can induce changes in cell morphology and motility . AKT inhibition cause by sodium selenate seems to be physiologically effective at low concentrations (Tsukamoto, Hama, Kogure, & Tsuchiya, 2013)
Theophylline
Only one study has identified Theophylline an activator of PP2A. The study also implies that PP2A activation is a way to control respiratory inflammation. The study was done in vitro with primary cultures of human airway smooth muscle cells (Patel et al., 2016).
Cellular reaction pathways
PP1 pathway
Stimulation of mammalian cells with TNFα increases intracellular C6 ceramide production, which in turn increases PP1 activity (Ghosh et al, 2007). It was previously thought that insulin was the primary stimulant of the PP1 pathway. However, it has now been shown that TNFα-mediated ceramide production increases the Ser/Thr phosphatase activity of PP1 while insulin does not, indicating a ceramide-specific response (Ghosh et al, 2007). Inhibitors of de novo ceramide synthesis seem to prevent PP1 activation (Ghosh et al, 2007). The effects of ceramide on insulin-stimulated glycogen synthase kinase 3β phosphorylation were abolished with PP1 inhibitors, further implicating that TNFα mediates its effects through a ceramide-activated PP1 which blocks insulin phosphorylation cascades involved in glycogen metabolism (Ghosh et al, 2007.
PP2A pathway
The generation of ceramide can cause a down-regulation of the c-myc gene, which will trigger a cellular cascade resulting in cell death through apoptotic mechanisms. In leukemia cell lines, CAPP is actively brought into cells for downstream regulation of the c-myc gene through ceramide-induced control (Wolff et al, 1994). Partial purification of a ceramide-activated PP2A indicated an inherent ability to dephosphorylate the antiapoptotic protein c-jun in vitro, suggesting that it could be a direct substrate for ceramide-activated PP2A (Ruvolo et al, 1999).
Ceramide specifically activates a mitochondrial PP2A which results in the prompt dephosphorylation and inactivation of Bcl2 (Ruvolo et al, 1999). Bcl2 is an anti-apoptotic protein that, when inactivated, can cause the cell to conduct apoptosis (Ruvolo et al, 1999). Regulation of Bcl2 is dependent upon the phosphorylation status of Ser70 (Janssens & Goris, 2001). This phosphorylated residue is directly responsible for the apoptotic mechanism of the protein and a dephosphorylation at this site with a CAPP will inhibit its activity (Janssens and Goris, 2001).
Induction of apoptosis in Jurkat cells increases PP2A activity due to the activation of caspase-3, and the subsequent cleavage of the scaffold subunit (Janssens & Goris, 2001). This increased activity can be observed in the decreased phosphorylation of MAPK pathway substrates (Janssens & Goris, 2001).
E4orf4, an inducer of apoptosis in transformed cells, interacts with PP2A (Janssens & Goris, 2001). Interaction can take place on either a Bα or B’ regulatory subunit (Janssens & Goris, 2001). Nevertheless, only interaction with the Bα subunit is sufficient for the induction of apoptosis in transformed cells (Janssens & Goris, 2001).
Related pathology
Alzheimer’s disease
Hyperphosphorylated Tau proteins dissociate from the microtubules to which they provided stability and are thought to polymerize into neurofibrillary tangles in the brain and contribute to the onset of Alzheimer’s disease (Janssens & Goris, 2001). The B subunit of a CAPP confers the ability to dephosphorylate hyperphosphorylated Tau proteins (Janssens & Goris, 2001). Hyperphosphorylated Tau can interact with the acidic face of the B subunit and allow the catalytic subunit to dephosphorylate the protein (Janssens & Goris, 2001). Treatment of neuronal cells with OA has been shown to cause Tau neurofibrillary tangles, indicating that disruptions of the interaction between CAPP, Tau and microtubules can lead to the onset of Alzheimer’s disease (Janssens & Goris, 2001).
Cancer
CAPP was first linked to carcinogenesis when it was noticed that OA acted as a tumor promoter and it was postulated that its inhibition of CAPP may confer this property (Janssens & Goris, 2001). The α and β isoforms of the scaffold subunit of CAPP have been identified as tumor suppressor genes in skin, lung, breast and colon-derived cell lines (Janssens & Goris, 2001). The B’ regulatory subunit of CAPP also appears to be overexpressed in malignant melanoma, as compared to regular epidermal cells (Janssens & Goris, 2001).
The B’ subunit seems to interact specifically with and dephosphorylate paxillin in the focal adhesions of cancer cells (Janssens & Goris, 2001). When truncated B’ γ subunits were expressed in melanoma cells, an increased rate of metastasis was observed (Janssens & Goris, 2001). The increased cell migration appears to be related to the increased phosphorylation of paxillin when dysfunctional B’ γ are expressed (Janssens & Goris, 2001).
In most primary human malignancies, telomerase is elevated, suggesting that telomerase is required for continuous cell division (Janssens & Goris, 2001). It has been shown that ceramide treatment can significantly reduce telomerase production in human lung carcinomas, indicating that CAPPs may be involved in counteracting uncontrolled cell growth (Ogretmen et al., 2001).
References
Chalfant, C., Kishikawa, K., Mumby, M., Kamibayashi, C., Bielawska, A., & Hannun, Y. (1999). Long chain ceramides activate protein phosphatase-1 and protein phosphatase-2A. Activation is stereospecific and regulated by phosphatidic acid. The Journal of Biological Chemistry, 274(29), 20313-7.
Egloff, Marie‐Pierre, Johnson, Deborah F., Moorhead, Greg, Cohen, Patricia T. W., Cohen, Philip, & Barford, David. (1997). Structural basis for the recognition of regulatory subunits by the catalytic subunit of protein phosphatase 1. EMBO Journal, 16(8), 1876-1887.
Ghosh, N., Patel, N., Jiang, K., Watson, J., Cheng, J., Chalfant, C., & Cooper, D. (2007). Ceramide-activated protein phosphatase involvement in insulin resistance via Akt, serine/arginine-rich protein 40, and ribonucleic acid splicing in L6 skeletal muscle cells. Endocrinology, 148(3), 1359-66.
Groves, Hanlon, Turowski, Hemmings, & Barford. (1999). The Structure of the Protein Phosphatase 2A PR65/A Subunit Reveals the Conformation of Its 15 Tandemly Repeated HEAT Motifs. Cell, 96(1), 99-110.
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Ogretmen, B., Kraveka, J., Schady, D., Usta, J., Hannun, Y., & Obeid, L. (2001). Molecular mechanisms of ceramide-mediated telomerase inhibition in the A549 human lung adenocarcinoma cell line. The Journal of Biological Chemistry, 276(35), 32506-14.
Patel, B. S., Rahman, M. M., Rumzhum, N. N., Oliver, B. G., Verrills, N. M., & Ammit, A. J. (2016). Theophylline represses IL-8 secretion from airway smooth muscle cells independently of phosphodiesterase inhibition novel role as a protein phosphatase 2A activator. American Journal of Respiratory Cell and Molecular Biology, 54(6), 792-801.
Ruvolo, P., Deng, X., Ito, T., Carr, B., & May, W. (1999). Ceramide induces Bcl2 dephosphorylation via a mechanism involving mitochondrial PP2A. The Journal of Biological Chemistry, 274(29), 20296-300.
Tan, X., Wright, D., Liu, S., Hovens, C., O'Brien, T., & Shultz, S. (2016). Sodium selenate, a protein phosphatase 2A activator, mitigates hyperphosphorylated tau and improves repeated mild traumatic brain injury outcomes. Neuropharmacology, 108, 382-393.
Tsukamoto, T., Hama, S., Kogure, K., & Tsuchiya, H. (2013). Selenate induces epithelial-mesenchymal transition in a colorectal carcinoma cell line by AKT activation. Experimental Cell Research, 319(13), 1913-1921.
Wang, X., Blanchard, J., Tung, Y., Grundke Iqbal, I., & Iqbal, K. (2015). Inhibition of protein phosphatase-2A (PP2A) by I1PP2A leads to hyperphosphorylation of tau, neurodegeneration, and cognitive impairment in rats. Journal of Alzheimer's Disease, 45(2), 423-435.
Wolff, R., Dobrowsky, R., Bielawska, A., Obeid, L., & Hannun, Y. (1994). Role of ceramide-activated protein phosphatase in ceramide-mediated signal transduction. The Journal of Biological Chemistry, 269(30), 19605-9.
Zhu, Y., Dong, A., Meyer, D., Pichon, O., Renou, J., Cao, K., & Shen, W. (2006). Arabidopsis NRP1 and NRP2 encode histone chaperones and are required for maintaining postembryonic root growth. The Plant Cell, 18(11), 2879-2892.