Ernest Y.C. Lee

A protein phosphatase-1-binding motif identified by the panning of a random peptide display library.

An unusually large number of regulatory or targeting proteins that bind to the catalytic subunit of protein phosphatase-1 have been recently reported. This can be explained by their possession of a common protein motif that interacts with a binding site on protein phosphatase-1. The existence of such a motif was established by the panning of a random peptide library in which peptide sequences are displayed on the Escherichia colibacterial flagellin protein for bacteria that bound to protein phosphatase-1. There were 79 isolates containing 46 unique sequences with the conserved motif VXF or VXW, whereX was most frequently His or Arg. In addition, this sequence was commonly preceded by 2–5 basic residues and followed by 1 acidic residue. This study demonstrates that binding to protein phosphatase-1 can be conferred to a protein by the presentation of a peptide motif on a surface loop. This binding motif is found in a number of protein phosphatase-1-binding proteins.

Activation of phosphorylase phosphatase by a novel procedure: Evidence for a regulatory mechanism involving the release of a catalytic subunit from enzyme-inhibitor complex(es) of higher molecular weight

Abstract Phosphorylase phosphatase in crude tissue extracts of rat liver, skeletal and heart muscle can be markedly activated by a treatment involving precipitation with ammonium sulfate and ethanol. The activation of the enzyme in rat liver extracts is shown to occur with the concomitant conversion of the enzyme from multiple molecular weight forms to a single form of lower molecular weight (M.W.∼30,000). After activation by ethanol treatment, phosphorylase phosphatase activities of rat liver, skeletal and heart muscle were shown by sucrose density ultracentrifugation to sediment in a similar manner with a sedimentation coefficient of 2.8S and M.W. 32,000.

A protein inhibitor of rabbit liver phosphorylase phosphatase

Abstract Extracts of rabbit liver contain a heat-stable, non-dialysable inhibitor of phosphorylase phosphatase. The inhibitory activity is destroyed by trypsin treatment or by ethanol precipitation. The kinetics of the inhibition are non-competitive with respect to phosphorylase a. The inhibitory activity is polydisperse on gel permeation chromatography. The mechanism of the inhibition is due to a direct interaction of the protein inhibitor with the enzyme.

Functional Roles of p12, the Fourth Subunit of Human DNA Polymerase δ

Mammalian DNA polymerase δ (pol δ), a key enzyme of chromosomal DNA replication, consists of four subunits as follows: the catalytic subunit; p125, which is tightly associated with the p50 subunit; p68, a proliferating cell nuclear antigen (PCNA)-binding protein; and a fourth subunit, p12. In this study, the functional roles of the p12 subunit of pol δ were studied. The inter-subunit interactions of the p12 subunit were determined by yeast two-hybrid assays and by pulldown assays. These assays revealed that p12 interacts with p125 as well as p50. This dual interaction of p12 suggests that it may serve to stabilize the p125-p50 interaction. p12 was shown to be a novel PCNA-binding protein. This was confirmed by identification of a PCNA-binding motif at its N terminus by binding assays and by site-directed mutagenesis. The activities and reaction products of recombinant pol δ containing a p12 mutant defective in PCNA binding, as well as purified recombinant pol δ and its subassemblies, were analyzed. Our results indicate that p12 contributes to PCNA-dependent pol δ activity, i.e. the p12-PCNA interaction is functional. Our data indicate that both p12 and p68 are required for optimal pol δ activity. This supports the hypothesis that the interaction between pol δ and PCNA is a divalent one that involves p12 and p68. We propose a model in which pol δ interacts with PCNA via at least two of its subunits, and one in which p12 could play a role in stabilizing the overall pol δ-PCNA complex as well as pol δ itself.

PCNA is ubiquitinated by RNF8

The ubiquitination of PCNA is an essential event in the operation of the DNA Damage Tolerance (DDT) pathway that is activated after DNA damage caused by UV or chemical agents during S-phase. This pathway allows the bypass of DNA damage by translesion synthesis that would otherwise cause replication fork stalling. PCNA is mono-ubiquitinated by Rad18-Rad6, and polyubiquitinated by Rad5-Ubc13/Uev1 in the DDT pathway. Mono-and polyubiquitination of PCNA are key processes in the translesion bypass and template switching sub-pathways of the DDT. DNA damage by IR causes DSBs, which trigger the DNA Damage Response (DDR). The ubiquitin ligase RNF8 has a critical role in the assembly of BRCA1 complexes at the DSBs in the DDR. We show that RNF8 readily mono-ubiquitinates PCNA in the presence of UbcH5c, and polyubiquitinates PCNA in the added presence of Ubc13/Uev1a. These reactions are the same as those performed by Rad18-Rad6 and Rad5-Ubc13. RNF8 depletion suppressed both UV and MNNG-stimulated mono-ubiquitination of PCNA, revealing that an RNF8-dependent pathway for PCNA ubiquitination is operative in vivo. These findings provide evidence that RNF8, a key E3 ligase in the DDR, may also play a role in the DDT.

A Novel DNA Damage Response RAPID DEGRADATION OF THE p12 SUBUNIT OF DNA POLYMERASE δ

Mammalian DNA polymerase (Pol) δ is essential for DNA replication. It consists of four subunits, p125, p50, p68, and p12. We report the discovery that the p12 subunit is rapidly degraded in cultured human cells by DNA damage or replication stress brought about by treatments with UV, methyl methanesulfonate, hydroxyurea, and aphidicolin. The degradation of p12 is due to an accelerated rate of proteolysis that is inhibited by the proteasome inhibitors, MG132 and lactacystin. UV treatment converts Pol δ in vivo to the three-subunit form lacking p12. This was demonstrated by its isolation using immunoaffinity chromatography. The three-subunit enzyme retains activity on poly(dA)/oligo(dT) templates but is impaired in its ability to extend singly primed M13 templates, clearly indicating that its in vivo functions are likely to be compromised. This transformation of Pol δ by modification of its quaternary structure is reversible in vitro by the addition of the p12 subunit and could represent a novel in vivo mechanism for the modulation of Pol δ function. UV and hydroxyurea-triggered p12 degradation is blocked in ATR–/– cells but not in ATM–/– cells, thereby demonstrating that p12 degradation is regulated by ATR, the apical kinase that regulates the damage response in S-phase. These findings reveal a novel addition to the cellular repertoire of DNA damage responses that also impacts our understanding of the role of Pol δ in both DNA replication and DNA repair.

Activation of Protein Phosphatase 1 FORMATION OF A METALLOENZYME

The recombinant catalytic subunit of protein phosphatase 1 is produced as an inactive enzyme which can be activated by Mn2+ (Zhang, Z., Bai, G., Deans-Zirattu, S., Browner, M. F., and Lee, E. Y. C.(1992) J. Biol. Chem. 267, 1484-1490). In this report, we have investigated the effects of divalent cations on the activity of recombinant catalytic subunit of protein phosphatase 1. Latent phosphatase 1 can be activated by Co2+ or Mn2+, whereas other metal ions tested including Fe2+, Zn2+, Mg2+, Ca2+, Cu2+, or Ni2+ were not effective or were only weakly effective in activating the enzyme. The Mn2+-stimulated activity was susceptible to inactivation by EDTA; however, the Co2+-activated phosphatase was stable after dilution and chelation of the Co2+ with excess EDTA. After stable activation of phosphatase 1 using 57Co2+, a stoichiometric amount of 57Co2+ was shown to be tightly bound to phosphatase 1. These findings demonstrate for the first time the generation of a stable metalloenzyme form of phosphatase 1. Fe2+ reversibly deactivated the Co2+-stimulated activity, but did not displace the bound Co2+. Interestingly, treatment of the enzyme with a combination of Fe2+ and Zn2+ (but not the individual metal ions) significantly activated phosphatase 1. These results suggest that at least two metal binding sites exist on the enzyme and that protein phosphatase 1 may be an iron/zinc metalloprotein in vivo.

Purification and properties of bovine myocardial phosphorylase phosphatase (protein phosphatase C).

Abstract Phosphorylase phosphatase was purified to homogeneity from bovine myocardium by the procedure of Brandt et al. ( Brandt, H., Capulong, Z. L., and Lee, E. Y. C., (1975 ) J. Biol. Chem. 250, 8038–8044). The purified enzyme consists of a single polypeptide chain of M r , 34,800 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The K m for phosphorylase a was 2.9 μ m and at V max , the enzyme had a turnover number of 11.7 mol phosphorylase a (dimer) converted/mol phosphatase/s. Phosphorylated histone and protamine were also substrates for this phosphatase. The K m for histone was 46 μ m (based on incorporated 32 P i and at V max a turnover number of 3.3 mol phosphate released/mol phosphatase/s was found. In general, the properties of the bovine phosphorylase phosphatase closely resemble those found for the rabbit liver enzyme.

Properties and regulation of liver phosphorylase phosphatase

Abstract A procedure was developed for the isolation of rabbit liver phosphorylase phosphatase. The properties of a preparation made by this procedure were examined. The enzyme had a molecular weight of 35,000, and was shown to behave as a protein phosphatase in that glycogen synthase and histones were also substrates. An activation phenomenon encountered during the development of the purification procedure was studied. Both ethanol and trypsin treatment lead to the activation and concomitant interconversion of enzyme activity in liver extracts from apparent multiple molecular weight forms to a single smaller form of M.W. 35,000. It was proposed that the enzyme exists in an inactive form(s) consisting of complexes of the M.W. 35,000 enzyme with protein inhibitors. The properties of a preparation of an endogenous rabbit liver protein inhibitor were examined and found to be consistent with this explanation. Furthermore, evidence was found for a single inactive form of the enzyme (M.W. 220,000) from which the multiple forms may be artifactually derived during homogenization. On the basis of these studies we have proposed a model for the relationship between the active and inactive forms of the enzyme, and the possible regulation of this interconversion. While a great deal of further investigation is needed to establish the true nature and regulation of the protein phosphatases, their elucidation is clearly fundamental to our understanding of the molecular mechanisms of the regulation of glycogen metabolism.

Liver protein phosphatases: Studies of the presumptive native forms of phosphorylase phosphatase activity in liver extracts and their dissociation to a catalytic subunit of Mr 35,000☆

Abstract Several aspects of the properties of phosphorylase phosphatase in crude rat liver extracts were investigated. Treatment of tissue extracts with either trypsin, ethanol, or urea was found to dissociate phosphorylase phosphatase activity to a form of M r 35,000. The M r 35,000 enzyme form was derived from three native enzyme forms. The major cytosolic form of phosphorylase phosphatase had a molecular weight of 260,000 as determined by gel filtration and was dissociated to a M r 35,000 form by treatment with either ethanol or urea. Treatment of the M r 260,000 form with trypsin led to its conversion to M r 225,000 and a M r 35,000 form. A minor cytosolic form of M r 200,000 was also present. This minor activity was latent until activated by trypsin treatment and was converted to a M r 35,000 form by such treatment. The third form was found to chromatograph as a form of molecular weight greater than 500,000 on gel filtration and, like the M r 200,000 form, was only detected after activation with trypsin. Subsequent to this treatment, it too behaved as a M r 35,000 enzyme. Although a single major enzyme form was present in the cytosol, multiple molecular weight forms could be generated in crude extracts simply by the use of vigorous mechanical homogenization procedures. This suggested that artifactual forms of enzyme may readily be produced, possibly by proteolytic cleavage of the native enzyme.