Carol MacKintosh

Cyanobacterial microcystin‐LR is a potent and specific inhibitor of protein phosphatases 1 and 2A from both mammals and higher plants

The cyclic heptapeptide, microcystin‐LR, inhibits protein phosphatases 1 (PP1) and 2A (PP2A) with K i, values below 0.1 nM. Protein phosphatase 2B is inhibited 1000‐fold less potently, while six other phosphatases and eight protein kinases tested are unaffected. These results are strikingly similar to those obtained with the tumour promoter okadaic acid. We establish that okadaic acid prevents the binding of microcystin‐LR to PP2A, and that protein inhibitors 1 and 2 prevent the binding of microcystin‐LR to PP1. We discuss the possibility that inhibition of PP1 and PP2A accounts for the extreme toxicity of microcystin‐LR, and indicate its potential value in the detection and analysis of protein kinases and phosphatases.

14-3-3-affinity purification of over 200 human phosphoproteins reveals new links to regulation of cellular metabolism, proliferation and trafficking.

14-3-3-interacting proteins were isolated from extracts of proliferating HeLa cells using 14-3-3 affinity chromatography, eluting with a phosphopeptide that competes with targets for 14-3-3 binding. The isolated proteins did not bind to 14-3-3 proteins (14-3-3s) after dephosphorylation with protein phosphatase 2A (PP2A), indicating that binding to 14-3-3s requires their phosphorylation. The binding proteins identified by tryptic mass fingerprinting and Western blotting include many enzymes involved in generating precursors such as purines (AMP, GMP and ATP), FAD, NADPH, cysteine and S-adenosylmethionine, which are needed for cell growth, regulators of cell proliferation, including enzymes of DNA replication, proteins of anti-oxidative metabolism, regulators of actin dynamics and cellular trafficking, and proteins whose deregulation has been implicated in cancers, diabetes, Parkinsonism and other neurological diseases. Several proteins bound to 14-3-3-Sepharose in extracts of proliferating cells, but not in non-proliferating, serum-starved cells, including a novel microtubule-interacting protein ELP95 (EMAP-like protein of 95 kDa) and a small HVA22/Yop1p-related protein. In contrast, the interactions of 14-3-3s with the N-methyl-D-aspartate receptor 2A subunit and NuMA (nuclear mitotic apparatus protein) were not regulated by serum. Overall, our findings suggest that 14-3-3s may be central to integrating the regulation of biosynthetic metabolism, cell proliferation, survival, and other processes in human cells.

Phosphorylation of serine 230 promotes inducible transcriptional activity of heat shock factor 1

Heat shock factor 1 (HSF1) is a serine‐rich constitutively phosphorylated mediator of the stress response. Upon stress, HSF1 forms DNA‐binding trimers, relocalizes to nuclear granules, undergoes inducible phosphorylation and acquires the properties of a transactivator. HSF1 is phosphorylated on multiple sites, but the sites and their function have remained an enigma. Here, we have analyzed sites of endogenous phosphorylation on human HSF1 and developed a phosphopeptide antibody to identify Ser230 as a novel in vivo phosphorylation site. Ser230 is located in the regulatory domain of HSF1, and promotes the magnitude of the inducible transcriptional activity. Ser230 lies within a consensus site for calcium/calmodulin‐dependent protein kinase II (CaMKII), and CaMKII overexpression enhances both the level of in vivo Ser230 phosphorylation and transactivation of HSF1. The importance of Ser230 was further established by the S230A HSF1 mutant showing markedly reduced activity relative to wild‐type HSF1 when expressed in hsf1−/− cells. Our study provides the first evidence that phosphorylation is essential for the transcriptional activity of HSF1, and hence for induction of the heat shock response.

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.

PRAS40 is a target for mammalian target of rapamycin complex 1 and is required for signaling downstream of this complex

Signaling through the mammalian target of rapamycin complex 1 (mTORC1) is positively regulated by amino acids and insulin. PRAS40 associates with mTORC1 (which contains raptor) but not mTORC2. PRAS40 interacts with raptor, and this requires an intact TOR-signaling (TOS) motif in PRAS40. Like TOS motif-containing proteins such as eIF4E-binding protein 1 (4E-BP1), PRAS40 is a substrate for phosphorylation by mTORC1. Consistent with this, starvation of cells of amino acids or treatment with rapamycin alters the phosphorylation of PRAS40. PRAS40 binds 14-3-3 proteins, and this requires both amino acids and insulin. Binding of PRAS40 to 14-3-3 proteins is inhibited by TSC1/2 (negative regulators of mTORC1) and stimulated by Rheb in a rapamycin-sensitive manner. This confirms that PRAS40 is a target for regulation by mTORC1. Small interfering RNA-mediated knockdown of PRAS40 impairs both the amino acid- and insulin-stimulated phosphorylation of 4E-BP1 and the phosphorylation of S6. However, this has no effect on the phosphorylation of Akt or TSC2 (an Akt substrate). These data place PRAS40 downstream of mTORC1 but upstream of its effectors, such as S6K1 and 4E-BP1.

Bioinformatic and experimental survey of 14-3-3-binding sites

More than 200 phosphorylated 14-3-3-binding sites in the literature were analysed to define 14-3-3 specificities, identify relevant protein kinases, and give insights into how cellular 14-3-3/phosphoprotein networks work. Mode I RXX(pS/pT)XP motifs dominate, although the +2 proline residue occurs in less than half, and LX(R/K)SX(pS/pT)XP is prominent in plant 14-3-3-binding sites. Proline at +1 is rarely reported, and such motifs did not stand up to experimental reanalysis of human Ndel1. Instead, we discovered that 14-3-3 interacts with two residues that are phosphorylated by basophilic kinases and located in the DISC1 (disrupted-in-schizophrenia 1)-interacting region of Ndel1 that is implicated in cognitive disorders. These data conform with the general findings that there are different subtypes of 14-3-3-binding sites that overlap with the specificities of different basophilic AGC (protein kinase A/protein kinase G/protein kinase C family) and CaMK (Ca2+/calmodulin-dependent protein kinase) protein kinases, and a 14-3-3 dimer often engages with two tandem phosphorylated sites, which is a configuration with special signalling, mechanical and evolutionary properties. Thus 14-3-3 dimers can be digital logic gates that integrate more than one input to generate an action, and coincidence detectors when the two binding sites are phosphorylated by different protein kinases. Paired sites are generally located within disordered regions and/or straddle either side of functional domains, indicating how 14-3-3 dimers modulate the conformations and/or interactions of their targets. Finally, 14-3-3 proteins bind to members of several multi-protein families. Two 14-3-3-binding sites are conserved across the class IIa histone deacetylases, whereas other protein families display differential regulation by 14-3-3s. We speculate that 14-3-3 dimers may have contributed to the evolution of such families, tailoring regulatory inputs to different physiological demands.

Tautomycin from the bacterium Streptomyces verticillatus: Another potent and specific inhibitor of protein phosphatases 1 and 2A

Tautomycin inhibited the catalytic subunits of protein phosphatase‐1 (K i app = 0.16 nM) more potently than protein phosphatase 2A (K i app = 0.4 nM), and the native forms of these enzymes in mammalian, protozoan and plant extracts were inhibited in a similar manner. Protein phosphatase 2B was inhibited 10 000‐fold less potently, while two other phosphatases and six protein kinases were unaffected at 10 μM. Okadaic acid prevented the binding of tautomycin to protein phosphatase 2A, indicating a common binding site for both inhibitors. The different relative potencies of tautomycin and okadaic acid for protein phosphatases 1 and 2A suggest that parallel use of both inhibitors may help to identify physiological substrates for each enzyme.

Complementary regulation of TBC1D1 and AS160 by growth factors, insulin and AMPK activators

AS160 (Akt substrate of 160 kDa) and TBC1D1 are related RabGAPs (Rab GTPase-activating proteins) implicated in regulating the trafficking of GLUT4 (glucose transporter 4) storage vesicles to the cell surface. All animal species examined contain TBC1D1, whereas AS160 evolved with the vertebrates. TBC1D1 has two clusters of phosphorylated residues, either side of the second PTB (phosphotyrosine-binding domain). Each cluster contains a 14-3-3-binding site. When AMPK (AMP-activated protein kinase) is activated in HEK (human embryonic kidney)-293 cells, 14-3-3s bind primarily to pSer237 (where pSer is phosphorylated serine) in TBC1D1, whereas 14-3-3 binding depends primarily on pThr596 (where pThr is phosphorylated threonine) in cells stimulated with IGF-1 (insulin-like growth factor 1), EGF (epidermal growth factor) and PMA; and both pSer237 and pThr596 contribute to 14-3-3 binding in cells stimulated with forskolin. In HEK-293 cells, LY294002 inhibits phosphorylation of Thr596 of TBC1D1, and promotes phosphorylation of AMPK and Ser237 of TBC1D1. In vitro phosphorylation experiments indicated regulatory interactions among phosphorylated sites, for example phosphorylation of Ser235 prevents subsequent phosphorylation of Ser237. In rat L6 myotubes, endogenous TBC1D1 is strongly phosphorylated on Ser237 and binds to 14-3-3s in response to the AMPK activators AICAR (5-aminoimidazole-4-carboxamide-1-b-D-ribofuranoside), phenformin and A-769662, whereas insulin promotes phosphorylation of Thr596 but not 14-3-3 binding. In contrast, AS160 is phosphorylated on its 14-3-3-binding sites (Ser341 and Thr642) and binds to 14-3-3s in response to insulin, but not A-769662, in L6 cells. These findings suggest that TBC1D1 and AS160 may have complementary roles in regulating vesicle trafficking in response to insulin and AMPK-activating stimuli in skeletal muscle.

Regulation of multisite phosphorylation and 14-3-3 binding of AS160 in response to IGF-1, EGF, PMA and AICAR.

AS160 (Akt substrate of 160 kDa) mediates insulin-stimulated GLUT4 (glucose transporter 4) translocation, but is widely expressed in insulin-insensitive tissues lacking GLUT4. Having isolated AS160 by 14-3-3-affinity chromatography, we found that binding of AS160 to 14-3-3 isoforms in HEK (human embryonic kidney)-293 cells was induced by IGF-1 (insulin-like growth factor-1), EGF (epidermal growth factor), PMA and, to a lesser extent, AICAR (5-aminoimidazole-4-carboxamide-1-b-D-ribofuranoside). AS160-14-3-3 interactions were stabilized by chemical cross-linking and abolished by dephosphorylation. Eight residues on AS160 (Ser318, Ser341, Thr568, Ser570, Ser588, Thr642, Ser666 and Ser751) were differentially phosphorylated in response to IGF-1, EGF, PMA and AICAR. The binding of 14-3-3 proteins to HA-AS160 (where HA is haemagglutinin) was markedly decreased by mutation of Thr642 and abolished in a Thr642Ala/Ser341Ala double mutant. The AGC (protein kinase A/protein kinase G/protein kinase C-family) kinases RSK1 (p90 ribosomal S6 kinase 1), SGK1 (serum- and glucocorticoid-induced protein kinase 1) and PKB (protein kinase B) displayed distinct signatures of AS160 phosphorylation in vitro: all three kinases phosphorylated Ser318, Ser588 and Thr642; RSK1 also phosphorylated Ser341, Ser751 and to a lesser extent Thr568; and SGK1 phosphorylated Thr568 and Ser751. AMPK (AMP-activated protein kinase) preferentially phosphorylated Ser588, with less phosphorylation of other sites. In cells, the IGF-1-stimulated phosphorylations, and certain EGF-stimulated phosphorylations, were inhibited by PI3K (phosphoinositide 3-kinase) inhibitors, whereas the RSK inhibitor BI-D1870 inhibited the PMA-induced phosphorylations. The expression of LKB1 in HeLa cells and the use of AICAR in HEK-293 cells promoted phosphorylation of Ser588, but only weak Ser341 and Thr642 phosphorylations and binding to 14-3-3s. Paradoxically however, phenformin activated AMPK without promoting AS160 phosphorylation. The IGF-1-induced phosphorylation of the novel phosphorylated Ser666-Pro site was suppressed by AICAR, and by combined mutation of a TOS (mTOR signalling)-like sequence (FEMDI) and rapamycin. Thus, although AS160 is a common target of insulin, IGF-1, EGF, PMA and AICAR, these stimuli induce distinctive patterns of phosphorylation and 14-3-3 binding, mediated by at least four protein kinases.

Purification of type 1 protein (serine/threonine) phosphatases by microcystin-Sepharose affinity chromatography

A microcystin (MC)‐Sepharose column was prepared by addition of 2‐aminoethanethiol to the α,β‐unsaturated carbonyl of the N‐methyldehydroalanine residue of MC‐LR, followed by reaction of the introduced amino group with N‐hydroxysuccinimide‐activated CH‐Sepharose. The MC‐Sepharose bound protein phosphatase‐1 (PPI) with high capacity and purified human PP1γ in one step from E. coli extracts. It was also used to purify forms of PPI bound to myofibrils from skeletal muscle. Two of these comprised PP1 completed to N‐terminal fragments of the M‐subunit which enhance its myosin phosphatase activity, while the third comprised PP1 and an N‐terminal fragment of the glycogen‐binding (G)‐subunit.