From the Experts

Kinases and Phosphatases: Structure and Functions Part I

Dr. Divyashree Nageswaran
Scientific Advisor
TGI ,United Kingdom

Protein phosphorylation is an ancient, universal, reversible and regulatory mechanism driven by the antagonistic activities of kinases and phosphatases. They constitute nearly 2-4% of the genes in any eukaryotic genome. Proteins are phosphorylated basically on 9 amino acids, including, tyrosine (Tyr), serine (Ser), threonine (Thr), cysteine (Cys), arginine (Arg), lysine (lys), aspartate (Asp), glutamate (Glu) and histidine (His). However, Ser, Thr and Tyr phosphorylation occurs predominantly in eukaryotes.

Protein phosphatases are a distinct superfamily of biological catalysts. These phosphatases are nonspecific and constitutively active “housekeeping” enzymes compared to kinase activities. Unlike protein kinases which are thousands in number, Arabidopsis thaliana (model plant/weed belongs to the family Brassicaceae) and Homo sapiens (humans) have nearly 150 phosphatases that form the core catalytic domains. Enzymes that dephosphorylate the major amino acid residues (Ser, Thr and Tyr) are classified into four major groups according to catalytic mechanism, domain sequence, structure and substrate specificity. Ser/Thr phosphatases form the large phosphoprotein phosphatase (PPP) family (PP1, PP2A, PP2B/PP3/calcineurin, PP4, PP5, PP6 and PP7) and a metallo-dependent (Mg2+ or Mn2+) protein phosphatase (PPM) family, involving PP2C and pyruvate dehydrogenase phosphatase.

PP2C is a highly conserved and nuclear-localized protein in mammalian genomes, involved in stress signalling, DNA damage checkpoint signalling, cell differentiation, proliferation, growth, survival, apoptosis and metabolism. There are 76 and 18 predicted PP2Cs (PPM) in Arabidopsis and humans. Few examples include the PP2Cα/PPM1A that acts as a SMAD phosphatase which terminates transforming growth factor-β and regulates bone morphogenetic protein signaling by dephosphorylation of SMAD proteins. PP2Cγ also acts as a pre-mRNA splicing factor and is an essential component of spliceosome assembly. PP2C/PPM1D dephosphorylates CHK-1 and p53 that are activated by DNA damage response ATM/ATR kinases.

Protein Tyr phosphatase (PTPs) superfamily is defined based on its catalytic signature CX5R. There are 107 genes in the human genome forming four members of the PTP family on the basis of protein structure, substrate specificity, and regulatory functions. Dual specificity phosphatases (DUSPs) consists of certain Ser/Thr phosphatases that dephosphorylate tyrosine residues or vice versa (e.g. Mitogen-activated protein kinase-phosphatase or MAPKP). PTPs and DUSPs are Cys-dependent enzymes that act as master regulators of cell signalling and their activities are directed towards mRNA, phosphoinositides, carbohydrate and many other phosphorylated molecules.

Aspartate-based phosphatases form the fourth group with an active site signature DxDx(V/T). This includes Haloacid dehalogenase family of phosphatases (e.g. EYA, chronophins), and transcription initiation factor IIF associating C-terminal domain (CTD) phosphatase-1 (FCP1)/ small CTD phosphatase, which dephosphorylate CTD of the largest subunit of Pol II. Contrast to protein kinases, each of these PPP families lack sequence and structural similarity with the remaining other three groups.

The Phosphoprotein phosphatase (PPP) family consists of 26 genes in Arabidopsis thaliana and 13 in Homo sapiens, respectively. All of its members contain three highly conserved sequence motifs within the 30 kD catalytic domain, such as GDxHG, GDxVDRG, and GNHE. The PP1 sub-family is well conserved across all eukaryotes with nine (TOPP 1-9) genes in Arabidopsis and three isoforms in humans, localized within the nucleus and cytosol. These TOPPs are predicted to function in regulation of cell cycle. This functional enzyme involves a catalytic (C) subunit and a regulatory (R) subunit. Over 200 known regulatory subunits bind directly with the PP1 catalytic domain (PP1c) through a small conserved motif RVXF. This interaction of PP1 with the specific R unit is critical for various cellular functions. For example, PP1 enzyme from Saccharomyces cerevisiae (budding yeast), encoded by GLC7 (PP1c) interacts with a meiosis-specific regulator GIP1 affects the transcription of late meiotic genes and is required for sporulation. Also, this GLC7/PP1 activity dephosphorylates 􀀀H2A and promotes recovery from DNA replication inhibition. The PPP1C interacts with its regulatory inhibitor subunit 2 (PPP1R2) for proper condensation of chromatin and maintenance of histone H3 phosphorylation during mouse oocyte meiosis. In Caenorhabditis elegans (nematode worm), a nucleoporin (MEL-28) docks the PP1c catalytic subunit to direct kinetochore- disassembly for proper chromosome segregation during oocyte meiosis I and nuclear reassembly during M-phase-interphase transition. A PTW/PP1 complex in humans regulate chromatin structure during mitosis transition.

PP2B, also known as “Calcineurin” is active in various calcium-dependent cellular processes, including signal transduction, immune response, neurodevelopment and memory, muscle development and cardiac hypertrophy. PP2B catalytic subunit consists of a N-terminal phosphatase domain and regulatory subunit B with a helical domain, calmodulin-binding motif and an autoinhibitory element. A few of these proteins are localized in the nucleus and its role is less known. Unlike humans, Arabidopsis lack PP2B (PP3) but was identified with four putative proteins belonging to PPKL enzyme family. PPKLs are PP1-related Ser/Thr phosphatases with a large N terminal kelch-like repeat domain and a C-terminal PP1-like domain. A novel quantitative trait locus (QTL) GL3.1 in rice, encodes a PPKL family, which controls grain size and yield by dephosphorylating the cell cycle regulator, Cyclin-T1;3.

PP5, PP7 and two other plant specific phosphatases form a separate clade excluding other PPP members. Unlike other PPPs, PP5 exhibits low activity in vitro and less similarity. They possess three N-terminal tetratricopeptide (TPR) motifs and C-terminal sequences, together act as autoinhibitory domains similar to PP7. PP5 is cytosol-nuclear localized in Arabidopsis, humans and yeast like other PPPs. In both plants and animals, PP5 enzymes are activated by arachidonic acid and polyunsaturated fatty acids that bind to TPR domains, and repressed by okadaic acid. Plant PP5s have notable roles in disease resistance, light detection and thermotolerance.

PP7 consists of a Ca2+ binding EF-hand domain, which binds to calmodulin in a calcium-dependent manner similar to PP2B. However, calmodulin inhibits AtPP7 unlike PP2B/calcineurins. There are two PP7s in humans and one in Arabidopsis. AtPP7 contains three inserts in the C-terminal half of its catalytic subunit. This PP7 enzyme can be activated by proteolytic cleavage, whose first insert in the catalytic domain act as an autoinhibitory region. Arabidopsis PP7 is exclusively nuclear localized and regulates blue-light signaling in plants.

The protein phosphatases PP2A, PP4 and PP6 are structurally related to each other yet have distinct biological roles. I’ll write about them in depth in the next issue.

Reference (Dec-20-A9)

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