Patricia T.W. Cohen

The structure of the tetratricopeptide repeats of protein phosphatase 5: implications for TPR-mediated protein-protein interactions.

The tetratricopeptide repeat (TPR) is a degenerate 34 amino acid sequence identified in a wide variety of proteins, present in tandem arrays of 3–16 motifs, which form scaffolds to mediate protein–protein interactions and often the assembly of multiprotein complexes. TPR‐containing proteins include the anaphase promoting complex (APC) subunits cdc16, cdc23 and cdc27, the NADPH oxidase subunit p67 phox, hsp90‐binding immunophilins, transcription factors, the PKR protein kinase inhibitor, and peroxisomal and mitochondrial import proteins. Here, we report the crystal structure of the TPR domain of a protein phosphatase, PP5. Each of the three TPR motifs of this domain consist of a pair of antiparallel α‐helices of equivalent length. Adjacent TPR motifs are packed together in a parallel arrangement such that a tandem TPR motif structure is composed of a regular series of antiparallel α‐helices. The uniform angular and spatial arrangement of neighbouring α‐helices defines a helical structure and creates an amphipathic groove. Multiple‐TPR motif proteins would fold into a right‐handed super‐helical structure with a continuous helical groove suitable for the recognition of target proteins, hence defining a novel mechanism for protein recognition. The spatial arrangement of α‐helices in the PP5–TPR domain is similar to those within 14‐3‐3 proteins.

Structural basis for the recognition of regulatory subunits by the catalytic subunit of protein phosphatase 1

The diverse forms of protein phosphatase 1 in vivo result from the association of its catalytic subunit (PP1c) with different regulatory subunits, one of which is the G‐subunit (GM) that targets PP1c to glycogen particles in muscle. Here we report the structure, at 3.0 Å resolution, of PP1c in complex with a 13 residue peptide (GM[63–75]) of GM. The residues in GM[63–75] that interact with PP1c are those in the Arg/Lys–Val/Ile–Xaa–Phe motif that is present in almost every other identified mammalian PP1‐binding subunit. Disrupting this motif in the GM[63–75] peptide and the M110[1–38] peptide (which mimics the myofibrillar targeting M110 subunit in stimulating the dephosphorylation of myosin) prevents these peptides from interacting with PP1. A short peptide from the PP1‐binding protein p53BP2 that contains the RVXF motif also interacts with PP1c. These findings identify a recognition site on PP1c, invariant from yeast to humans, for a critical structural motif on regulatory subunits. This explains why the binding of PP1 to its regulatory subunits is mutually exclusive, and suggests a novel approach for identifying the functions of PP1‐binding proteins whose roles are unknown.

5′-AMP inhibits dephosphorylation, as well as promoting phosphorylation, of the AMP-activated protein kinase. Studies using bacterially expressed human protein phosphatase-2Cα and native bovine protein phosphatase-2Ac

Human protein phosphatase‐2Cα (PP2Cα) was purified to homogeneity after expression in Escherichia coli. AMP inhibited the dephosphorylation of AMP‐activated protein kinase (AMPK), but not phosphocasein, by PP2Cα. The concentration dependence and the effects of other nucleotides (ATP and formycin A‐5′‐monophosphate) suggest that AMP acts by binding to the same site which causes direct allosteric activation of AMPK. A similar, although less pronounced, effect was observed with another protein phosphatase (PP2Ac). We have now shown that AMPK activates the AMPK cascade by four mechanisms, which should make the system exquisitely sensitive to changes in AMP concentration.

Identification of the Ca2+‐dependent modulator protein as the fourth subunit of rabbit skeletal muscle phosphorylase kinase

Kakuichi et al. [l] and Cheung [2,3] were the first to demonstrate the presence of a factor in brain homogenates, which in the presence of Ca”‘, stimulated the activity of one of the cyclic nucleotide phosphodiesterases of this tissue. This factor was subsequently shown to be a small heat stable calcium binding protein, which was present in high concentrations in a wide variety of animal tissues [4-61. Following its purification to apparent homogeneity from bovine brain [7] and bovine heart [8] , it was noted that its physico-chemical properties were very similar to the calcium-binding subunit of rabbit skeletal muscle troponin, troponln-C, the protein which confers calcium sensitivity to actomyosin ATPase [9,10] . This idea was substantiated by the determination of the amino acid sequence of the ‘calcium-dependent modulator’ from bovine brain [ 1 l] and rat testis [ 121, which showed extensive homology with troponin-C, and by the finding that the ‘modulator’ could substitute for troponin-C in restoring calcium sensitivity to actomyosin ATPase in reconstituted systems [ 131. Troponin-C can also substitute for the ‘modulator’ in the activation of cyclic nucleotide

A novel human protein serine/threonine phosphatase, which possesses four tetratricopeptide repeat motifs and localizes to the nucleus.

A novel human protein serine/threonine phosphatase, PP5, and a structurally related phosphatase in Saccharomyces cerevisiae, PPT1, have been identified from their cDNA and gene respectively. Their predicted molecular mass is 58 kDa and they comprise a C‐terminal phosphatase catalytic domain and an N‐terminal domain, which has four repeats of 34 amino acids, three of which are tandemly arranged. The phosphatase domain possesses all the invariant motifs of the PP1/PP2A/PP2B gene family, but is not closely related to any other known member (< or = 40% identity). Thus PP5 and PPT1 comprise a new subfamily. The repeats in the N‐terminal domain are similar to the tetratricopeptide repeat (TPR) motifs which have been found in several proteins that are required for mitosis, transcription and RNA splicing. Bacterially expressed PP5 is able to dephosphorylate serine residues in proteins and is more sensitive than PP1 to the tumour promoter okadaic acid. A 2.3 kb mRNA encoding PP5 is present in all human tissues examined. Investigation of the intracellular distribution of PP5 by immunofluorescence, using two different antibodies raised against the TPR and phosphatase domains, localizes PP5 predominantly to the nucleus. This suggests that, like other nuclear TPR‐containing proteins, it may play a role in the regulation of RNA biogenesis and/or mitosis.

The cyanobacterial toxin microcystin binds covalently to cysteine-273 on protein phosphatase 1

The interaction between protein phosphatase 1 (PP1) and microcystin (MC) was stable in 1% SDS or 70% formic acid indicative of a covalent interaction. Here we isolate the MC‐binding peptide and demonstrate that Cys273 of PP1 binds covalently to the methyl‐dehydroalanine (Mdha) residue of the toxin. Mutation of Cys273 to Ala, Ser or leu abolished covalent binding to MC, as did reduction of the Mdha residue of the toxin with ethanethiol. The abolition of covalent binding increased the IC50 for toxin inhibition of PP1 by 5‐ to 20‐fold. The covalent binding of MC to protein serine/threonine phosphatases explains the failure to detect this toxin post‐mortem in suspected cases of MC poisoning.

Protein serine/threonine phosphatases; an expanding family

Five protein serine/threonine phosphatases (PP) have been identified by cloning cDNA from mammalian and Drosophila libraries. These novel enzymes, which have not yet been detected by the techniques of protein chemistry and enzymology, are termed PPV, PP2Bw, PPX, PPY and PPZ. The complete amino acid sequences of PPX, PPY and PPZ and an almost complete sequence of PPV are presented. In the catalytic domain PPV and PPX are more similar to PP2A (57–69% identity) than PPI (45–49% identity), while PPY and PPZ are more similar to PP1 (66–68% identity) than PP2A (44% identity). The cDNA for PP2BW encodes a novel Ca2+/calmodulin‐dependent protein phosphatase only 62% identical to PP2B in the catalytic domain. Approaches for determining the cellular functions of these protein phosphatases are discussed.

Molecular basis for TPR domain‐mediated regulation of protein phosphatase 5

Protein phosphatase 5 (Ppp5) is a serine/threonine protein phosphatase comprising a regulatory tetratricopeptide repeat (TPR) domain N‐terminal to its phosphatase domain. Ppp5 functions in signalling pathways that control cellular responses to stress, glucocorticoids and DNA damage. Its phosphatase activity is suppressed by an autoinhibited conformation maintained by the TPR domain and a C‐terminal subdomain. By interacting with the TPR domain, heat shock protein 90 (Hsp90) and fatty acids including arachidonic acid stimulate phosphatase activity. Here, we describe the structure of the autoinhibited state of Ppp5, revealing mechanisms of TPR‐mediated phosphatase inhibition and Hsp90‐ and arachidonic acid‐induced stimulation of phosphatase activity. The TPR domain engages with the catalytic channel of the phosphatase domain, restricting access to the catalytic site. This autoinhibited conformation of Ppp5 is stabilised by the C‐terminal αJ helix that contacts a region of the Hsp90‐binding groove on the TPR domain. Hsp90 activates Ppp5 by disrupting TPR–phosphatase domain interactions, permitting substrate access to the constitutively active phosphatase domain, whereas arachidonic acid prompts an alternate conformation of the TPR domain, destabilising the TPR–phosphatase domain interface.

Comparison of the specificities of p70 S6 kinase and MAPKAP kinase-1 identifies a relatively specific substrate for p70 S6 kinase: the N-terminal kinase domain of MAPKAP kinase-1 is essential for peptide phosphorylation.

xxR/KxRxxSxx sequences were phosphorylated with high efficiency by both p70 S6 kinase (p70S6K) and MAPKAP kinase‐1. The best substrate for MAPKAP kinase‐1 (KKKNRTLSVA) was phosphorylated with a K m of 0.17 μM, and the best substrate for p70S6K (KKRNRTLSVA) with a K m of 1.5 μM. The requirement of both enzymes for Arg/Lys at position n‐5 could be partially replaced by inserting basic residues at other positions, especially by an Arg at n ‐ 2 or n ‐ 4. MAPKAP kinase‐1 (but not p70S6K) tolerated lack of any residue at n ‐ 5 if Arg was present at n ‐ 2 and n ‐ 3. p70S6K (but not p90S6K) tolerated Thr at position n and absence of any residue at n + 2. The peptide KKRNRTLTV, which combined these features, was relatively selective for p70S6K having a 50‐fold higher V max/K m than MAPKAP kinase‐1. Inactivation of the N‐terminal kinase domain of MAPKAP kinase‐1, which is 60% identical to p70S6K, abolished activity towards all peptides tested, but the enzyme retained 30–40% of its activity if the C‐terminal kinase domain was inactivated.

Isolation and sequence analysis of a cDNA clone encoding a type-1 protein phosphatase catalytic subunit: Homology with protein phosphatase 2A

A 1.5 kb clone containing the full‐length coding sequence of a type‐1 protein phosphatase catalytic subunit has been isolated from a rabbit skeletal muscle cDNA library constructed in λgt10. The protein sequence deduced from the cDNA contains 311 residues and has a molecular mass of 35.4 kDa. A single mRNA species at 1.6 kb was visualized by Northern blotting. The type‐1 protein phosphatase was strikingly homologous to protein phosphatase 2A, 49% of the amino acids between residues 11 and 280 being identical. The first 10 and last 31 residues were dissimilar. Residues 1–101 of the type‐1 protein phosphatase also showed 21% sequence identity with a region of mammalian alkaline phosphatases.