Richard E. Honkanen

Regulators of Serine / Threonine Protein Phosphatases at the Dawn of a Clinical Era?

Reversible phosphorylation is a key mechanism for regulating the biological activity of many human proteins that affect a diverse array of cellular processes, including protein-protein interactions, gene transcription, cell-cycle progression and apoptosis. Once viewed as simple house keeping enzymes, recent studies have made it eminently clear that, like their kinase counterparts, protein phosphatases are dynamic and highly regulated enzymes. Therefore, the development of compounds that alter the activity of specific phosphatases is rapidly emerging as an important area in drug discovery. Because >98% of protein phosphorylation occurs on serine and threonine residues, the identification of agents that alter the activity of specific serine/threonine phosphatases seems especially promising for drug development in the future. This review is focused on the enzymes encoded by the PPP-gene family, which includes PP1, PP2A, PP2B, PP4, PP5, PP6 and PP7. The structure/functions of human phosphatases will be addressed briefly, as will the natural product inhibitors of PP1-PP6 (e.g. okadaic acid, microcystins, nodularin, cantharidin, calyculin A, tautomycin, and fostriecin). The development of chimeric antisense oligonucleotides that support RNAase H mediated degradation of the targeted mRNA has resulted in compounds capable of specifically suppressing the expression of PP5 (ISIS 15534) and PP1gamma 1 (ISIS 14435) in human cells. Such compounds have already proven useful for the validation of drug targets, and if difficulties associated with systemic delivery of antisense oligonucleotides can be overcome, antisense is poised to have a major impact on the clinical management of many human disorders.

Cantharidin, another natural toxin that inhibits the activity of serine/threonine protein phosphatases types 1 and 2A

Cantharidin, a natural toxicant of blister beetles, is a strong inhibitor of protein phosphatases types 1(PP1) and 2A (PP2A). Like okadaic acid, cantharidin inhibits the activity of the purified catalytic subunit of PP2A (IC50 = 0.16 μM) at a lower concentration than that of PPI (IC50 = 1.7 μM) and only inhibits the activity of protein phosphatase type 2B (PP2B) at high concentrations. Dose‐inhibition studies conducted with whole cell homogenates indicate that cantharidin also inhibits the native forms of these enzymes. Thus, cantharidin, which is economical and readily available, may be useful as an additional probe for studying the functions of serine/threonine protein phosphatases.

Small-molecule inhibitors of ser/thr protein phosphatases: specificity, use and common forms of abuse.

Natural product extracts have proven to be a rich source of small molecules that potently inhibit the catalytic activity of certain PPP-family ser/thr protein phosphatases. To date, the list of inhibitors includes okadaic acid (produced by marine dinoflagelates, Prorocentrum sp. and Dinophysis sp.), calyculin A, dragmacidins (isolated from marine sponges), microcystins, nodularins (cyanobacteria, Microcystis sp. and Nodularia sp.), tautomycin, tautomycetin, cytostatins, phospholine, leustroducsins, phoslactomycins, fostriecin (soil bacteria, Streptomyces sp.), and cantharidin (blister beetles, approx 1500 species). Many of these compounds share structural similarities, and several have become readily available for research purposes. Here we will review the specificity of available inhibitors and present methods for their use in studying sensitive phosphatases. Common mistakes in the employment of these compounds will also be addressed briefly, notably the widespread misconception that they only inhibit the activity of PP1 and PP2A. Inhibitors of PP2B (calcineurin) will only be mentioned in passing, except to state that, in our hands, cypermethrin, deltamethrin, and fenvalerate, which are sold as potent inhibitors of PP2B, do not inhibit the catalytic activity of PP2B.

Fostriecin, an antitumor antibiotic with inhibitory activity against serine/threonine protein phosphatases types 1 (PP1) and 2A (PP2A), is highly selective for PP2A

Fostriecin, an antitumor antibiotic produced by Streptomyces pulveraceus, is a strong inhibitor of type 2A (PP2A; IC50 3.2 nM) and a weak inhibitor of type 1 (PP1; IC50 131 μM) serine/threonine protein phosphatases. Fostriecin has no apparent effect on the activity of PP2B, and dose‐inhibition studies conducted with whole cell homogenates indicate that fostriecin also inhibits the native forms of PP1 and PP2A. Studies with recombinant PP1/PP2A chimeras indicate that okadaic acid and fostriecin have different binding sites.

Serine/threonine protein phosphatase type 5 acts upstream of p53 to regulate the induction of p21(WAF1/Cip1) and mediate growth arrest

Understanding how alterations in growth control pathways are translated into changes in the cell cycle regulatory machinery is a major challenge for understanding the development of human cancers. The ability of both tumor suppressor proteins, p53 and BRCA1, to induce the expression of p21WAF1/Cip1 in combination with the inhibitory activity of p21WAF1/Cip1 against cyclin-dependent kinases suggests that the regulation of p21WAF1/Cip1 expression is an important aspect of mammalian cell cycle growth control. To elucidate the role of serine/threonine protein phosphatase type 5 (PP5) in processes regulating cell cycle progression, we developed antisense oligodeoxynucleotides targeted against PP5 (e.g. ISIS 15534) that specifically inhibit PP5 gene expression. Employing ISIS 15534, we demonstrate that the specific inhibition of PP5 gene expression has a marked antiproliferative effect on cells, characterized by induction of p21WAF1/Cip1 and the subsequent arrest of cell growth. Investigations into the mechanisms leading to growth arrest reveal that, in the absence of PP5, the expression of p21WAF1/Cip1 is induced in p53-competent A549 cells but not in p53 protein-deficient T-24 cells. Employing a stable cell line derived from p53-deficient human fibroblast that contains tetracycline-regulated transactivator and operator plasmids to control the expression of wild-type p53 (TR9-7 cells), we then show that the induction of p21WAF1/Cip1, which occurs in response to the inhibition of PP5 expression, requires the p53 protein. Additional studies indicate that PP5 acts upstream of p53, influencing both the phosphorylation state and the ability of p53 to bind DNA, without causing an increase in p53 gene transcription. Together these studies suggest that PP5 is a regulatory component of a signaling pathway that affords replicating cells G1 checkpoint growth control and that it is the regulation of PP5 that, in turn, controls p53-mediated expression of p21WAF1/Cip1 and growth arrest in this pathway. In addition, since the inhibition of PP5 gene expression has marked antiproliferative activity and the overexpression of p21WAF1/Cip1 blocks the growth of tumor cells, these studies suggest that compounds that inhibit of PP5 gene expression may be useful in the treatment of human cancers.

Molecular Cloning, Expression, and Characterization of a Novel Human Serine/Threonine Protein Phosphatase, PP7, That Is Homologous toDrosophila Retinal Degeneration C Gene Product (rdgC)

A novel serine/threonine protein phosphatase (PPase) designated PP7 was identified from cDNA produced from human retina RNA. PP7 has a molecular mass of ∼75 kDa, and the deduced amino acid sequence of PP7 contains a phosphatase catalytic core domain that possesses all of the invariant motifs of the PP1, PP2A, PP2B, PP4, PP5, and PP6 gene family. However, PP7 has unique N- and C-terminal regions and shares <35% identity with the other known PPases. The unique C-terminal region of PP7 contains multiple Ca2+ binding sites (i.e. EF-hand motifs). This region of PP7 is similar to the Drosophila retinal degeneration C gene product (rdgC), and PP7 and rdgC share 42.1% identity. Unlike the other known PPases, the expression of PP7 is not ubiquitous; PP7 was only detected in retina and retinal-derived Y-79 retinoblastoma cells. Expression of recombinant human PP7 in baculovirus-infected SF21 insect cells produces an active soluble enzyme that is capable of utilizing phosphohistone and p-nitrophenyl phosphate as substrates. The activity of recombinant PP7 is dependent on Mg2+ and is activated by calcium (IC50 ≅ 250 μm). PP7 is not affected by calmodulin and is insensitive to inhibition by okadaic acid, microcystin-LR, calyculin A, and cantharidin.

Serine / threonine protein phosphatase 5 (PP5) participates in the regulation of glucocorticoid receptor nucleocytoplasmic shuttling

BackgroundIn most cells glucocorticoid receptors (GR) reside predominately in the cytoplasm. Upon hormone binding, the GR translocates into the nucleus, where the hormone-activated GR-complex regulates the transcription of GR-responsive genes. Serine/threonine protein phosphatase type 5 (PP5) associates with the GR-heat-shock protein-90 complex, and the suppression of PP5 expression with ISIS 15534 stimulates the activity of GR-responsive reporter plasmids, without affecting the binding of hormone to the GR.ResultsTo further characterize the mechanism by which PP5 affects GR-induced gene expression, we employed immunofluorescence microscopy to track the movement of a GR-green fluorescent fusion protein (GR-GFP) that retained hormone binding, nuclear translocation activity and specific DNA binding activity, but is incapable of transactivation. In the absence of glucocorticoids, GR-GFP localized mainly in the cytoplasm. Treatment with dexamethasone results in the efficient translocation of GR-GFPs into the nucleus. The nuclear accumulation of GR-GFP, without the addition of glucocorticoids, was also observed when the expression of PP5 was suppressed by treatment with ISIS 15534. In contrast, ISIS 15534 treatment had no apparent effect on calcium induced nuclear translocation of NFAT-GFP.ConclusionThese studies suggest that PP5 participates in the regulation of glucocorticoid receptor nucleocytoplasmic shuttling, and that the GR-induced transcriptional activity observed when the expression of PP5 is suppressed by treatment with ISIS 15534 results from the nuclear accumulation of GR in a form that is capable of binding DNA yet still requires agonist to elicit maximal transcriptional activation.

A low voltage-activated Ca2+ current mediates cytokine-induced pancreatic β-cell death

Insulin-dependent diabetes mellitus is characterized by the selective destruction of pancreatic β-cells. Chronic treatment with cytokines induced a low voltage-activated (LVA) Ca2+ current in mouseβ -cells. The concomitant increase in the basal cytoplasmic free Ca2+ concentration ([Ca2+]i) was associated with DNA fragmentation and cell death. Antagonists of LVA Ca2+ channels prevented this elevation of basal[ Ca2+]i and DNA fragmentation and reduced the percentage of cell death. Exposure to cytokines did not affect the profile of Ca2+ currents or basal[ Ca2+]i in glucagon-secreting α-cells. An increased Ca2+ signal through LVA Ca2+ channels may thus be a key feature in cytokine-induced β-cell destruction.

Label-free quantitative proteomics and SAINT analysis enable interactome mapping for the human Ser/Thr protein phosphatase 5

Affinity purification coupled to mass spectrometry (AP‐MS) represents a powerful and proven approach for the analysis of protein–protein interactions. However, the detection of true interactions for proteins that are commonly considered background contaminants is currently a limitation of AP‐MS. Here using spectral counts and the new statistical tool, Significance Analysis of INTeractome (SAINT), true interaction between the serine/threonine protein phosphatase 5 (PP5) and a chaperonin, heat shock protein 90 (Hsp90), is discerned. Furthermore, we report and validate a new interaction between PP5 and an Hsp90 adaptor protein, stress‐induced phosphoprotein 1 (STIP1; HOP). Mutation of PP5, replacing key basic amino acids (K97A and R101A) in the tetratricopeptide repeat (TPR) region known to be necessary for the interactions with Hsp90, abolished both the known interaction of PP5 with cell division cycle 37 homolog and the novel interaction of PP5 with stress‐induced phosphoprotein 1. Taken together, the results presented demonstrate the usefulness of label‐free quantitative proteomics and statistical tools to discriminate between noise and true interactions, even for proteins normally considered as background contaminants.

Serine/Threonine Protein Phosphatase Type 1γ1 Is Required for the Completion of Cytokinesis in Human A549 Lung Carcinoma Cells

In lower eukaryotic organisms, the loss of serine/threonine protein phosphatase type 1 (PP1) results in growth arrest after the onset of mitosis. In humans, four highly homologous isoforms of PP1 (PP1α, PP1δ, PP1γ1, and PP1γ2) have been identified. Determining the roles of these phosphatases, however, has proven difficult due to the lack of subtype-specific inhibitors. In this study, we developed chimeric antisense 2′-O-(2-methoxy)ethylphosphothioate oligonucleotides targeting human PP1γ1 that specifically inhibit PP1γ1 gene expression. Two potent antisense oligonucleotides (ISIS 14435 and 14439; IC50 ∼ 50 nm) were then employed to elucidate the cellular functions of PP1γ1 during cell cycle progression. In A549 cells, the inhibition of PP1γ1 expression resulted in a dose-dependent inhibition of cellular proliferation, with growth arrest occurring after ∼36–48 h, when PP1γ1 mRNA expression was inhibited by >85%. Fluorescence-activated cell sorter analysis revealed that ISIS 14435/14439-induced growth arrest was associated with an increase in the number of cells containing 4N DNA. Immunostaining of treated cells revealed that the inhibition of PP1γ1 expression had no apparent effect on the formation of mitotic spindles. However, decreased expression was associated with the failure of cell division in a late stage of cytokinesis and the formation of dikaryons.