DR. DIVYASHREE NAGESWARAN
TGI, Scientific Advisor
This is a continuation to my article featured in the December 2020 Issue. As promised in the last edition, in this article I am going to introduce the protein structure and highlight key functions of PP2A, PP4 and PP6 phosphatases in humans, Arabidopsis and other model systems. Another point I stressed about earlier was that these phosphatases are structurally related to each other and that their catalytic domains are conserved across eukaryotes.
PP2A is a holoenzyme that comprises a catalytic (C), scaffolding (A) and regulatory (B) subunits. There are 5 catalytic isoforms in Arabidopsis (C1, C2, C3, C4 and C5) and 2 (α and β) in humans. The catalytic subunits of PP2A, PP4 and PP6 are indirectly associated with regulatory (R or B) subunits using scaffold (A) domains as heterotrimers, except PP1. Both Arabidopsis and human PP2A subunit A are 3 (A1/RCN1, A2 and A3 or PR65 family) in number, comprising HEAT tandem repeats to bind C and B subunits together. These catalytic domains undergo reversible methylation, a covalent post-translational modification at the C-terminal which controls their interactions with regulatory subunits. Methylation of catalytic domains are catalysed by key activating and inactivating proteins (TAP46, TIP41, PTPA, LCMT1 and PME-1) that are present across eukaryotes. PP2A regulatory B subunits are classified into structurally different proteins, such as B/B55, B’/B56, B’’/B72/EF-hand and B’’’/B93/Striatin domains that determine their substrate specificity and sub-cellular localization. Arabidopsis contains 2 B, 9 B’ and 6 B’’ subunits, whereas humans have 4 B, 5 B’’, 4 B’’ and 3 B’’’ subunits, respectively. Both A and C subunits are highly concentrated in the nucleus, whereas certain B subunits are enriched either in the cytosol or nucleus.
PP2As regulate a variety of biological processes such as DNA replication, transcription and translation, post-translational modifications, signal transduction, apoptosis and cell proliferation. Particularly, human PP2A complex dephosphorylates p53 and many other critical cancer molecules, emphasizing its role as a “tumor suppressor”. Plant PP2As majorly participate in stress signalling pathways and nuclear functions. For example, Arabidopsis TAP46, a PP2A associated protein regulates cold stress signalling. Arabidopsis histone deacetylase 14 (HDA14) acts as a B regulatory subunit, which can deacetylate α-tubulin by direct association with PP2A-A subunit enriched in the microtubules with the histone acetyltransferase ELP3. Relevant to meiosis, PP2A forms a complex with Shugoshin (Sgo1/MEI-S332) and protects centromeric sister chromatid cohesion from separase during meiosis I in S. cerevisiae. Similarly, PP2AB’α and β from Arabidopsis specifically localize to centromeres until metaphase II, suggesting a role for the PP2A complex in centromeric cohesion during meiosis.
PP4/PPP4C/PPX in humans (HsPP4) exist either in heterodimers or heterotrimers and are predominantly localized to centrosomes and nucleus. The catalytic domain PP4 interacts with regulatory subunits PP4R1-PP4R4 or PP4R2-PP4R3α/β. The two regulatory subunits PP4R1 and PP4R4 carry HEAT repeat domains as that of PP2A scaffolding (A) subunits. However, PP4R1 and PP4R4 partners with PP4C and not with PP2A catalytic subunit. Similarly, yeast PPH3/PP4 forms a complex with PSY4/R2 and PSY2/R3 regulatory domains in budding yeast that is conserved similar to human PP4-PP4R2-PP4R3α/β complex. Both R2 and R3 subunits in yeast are nuclear-localized, whereas PPH3 localization is nuclear cytoplasmic.
PP4 is vital for cellular signalling and nuclear-related processes as that of PP2A. A PPH3/PP4 phosphatase complex dephosphorylates Tel1/Mec1 (ATM/ATR) kinases-mediated γH2AX and controls DNA damage checkpoint recovery. HsPP4 also acts as a γH2AX phosphatase important for DNA damage recovery. Similar to yeast, HsPP4 interacts with PP4R2-PP4R3β to specifically dephosphorylate ATR-mediated γH2AX generated during DNA replication. The PP4C-PP4R2 complex in human cells dephosphorylates the phospho-replication factor RPA-2 to facilitate DSB repair via homologous recombination. Yeast PPH3/PP4 is required for DSB repair by homologous recombination coupled with termination of DNA damage checkpoint signalling. The PPH3-PSY2 interaction is involved in regulating efficient non-homologous end joining (NHEJ) pathway, DNA repair and cell cycle progression. With reference to meiosis, a Mec1 kinase-PPH3 phosphatase-dependent checkpoint machinery mediated by substrate Zip1 stabilizes centromeric pairing in response to meiotic recombination initiation, thus connects meiotic chromosome dynamics to DSB repair.
Like in human cells, PP4 is centrosome-localized in Drosophila and C.elegans. This enzyme is required for microtubule organisation at centrosomes in Drosophila embryos. In C.elegans, PP4 is encoded by two genes PPH-4.1 and PPH-4.2. PPH4.1 is essential for mitotic spindle formation at centrosomes and sperm meiosis. During oogenesis, PPH4.1 is central for chiasmata formation after pairing and crossing over during meiotic prophase I. Also, PPH4.1 has numerous roles in meiotic prophase chromosome dynamics and contributes to maintenance of crossover competence with aging in the germ lines.
Arabidopsis PP4/PPX comprises two catalytic isoforms, PPX-1 and PPX-2, which may be localized in the nucleus, cytoplasm and plastids. PPX-1 and PPX-2 genes comprise eight exons and seven introns that are expressed at very low levels in flowers, leaves, stems and roots. This intron-exon organization of PP4/PPX is entirely different from PP2A family, although structurally related. AtPP4/PPX interact with regulatory subunits, including PP4R2 and PP4R3/SMEK1. This PP4R2 may function similar to human PP4R2 and yeast PSY4/R2. AtPP4 and PP4R3/SMEK1 (suppressor of MEK1) complex is shown to dephosphorylate HYL1 (Hyponastic Leaves 1), a core co-factor which promotes miRNA biogenesis by antagonizing the MAPK (Mitogen-activated protein kinase) cascade. Plant PP4 is predicted to function in DNA damage repair/sensitivity, as identified in humans and yeast.
PP6 catalytic subunit in humans interact with three regulatory ankyrin-repeat domains (ANKRD28, ANKRD44 and ANKRD52) and also with a conserved set of binding partners called Sit4-associated proteins (SAPS), PP6R1, PP6R2 and PP6R3. Human PP6, yeast Sit4 and PPV in Drosophila melanogaster regulate various biological processes, such as transcription, translation, cell cycle and morphogenesis. Arabidopsis PP6 is encoded by two genes, including AtFyPP3 and AtFyPP1/AtPP6 that are localized in the cytosol and bind to regulatory subunits, referred as SAL domains (SAL1, SAL2, SAL3 and SAL4). These FyPP/PP6 proteins have diverse roles in phytochrome-associated light regulation, flowering time control, PIN phosphorylation and auxin efflux as well as abscisic acid (ABA) signalling.
In my next article, I would pick a phosphatase and elucidate its role in the context of a specific mechanism in cells, say “DNA repair and recombination”.