Prognostic value of MACC1 and proficient mismatch repair status for recurrence risk prediction in stage II colon cancer patients: the BIOGRID studies.

BACKGROUND: We assessed the novel MACC1 gene to further stratify stage II colon cancer patients with proficient mismatch repair (pMMR). PATIENTS AND METHODS: Four cohorts with 596 patients were analyzed: Charité 1 discovery cohort was assayed for MACC1 mRNA expression and MMR in cryo-preserved tumors. Charité 2 comparison cohort was used to translate MACC1 qRT-PCR analyses to FFPE samples. In the BIOGRID 1 training cohort MACC1 mRNA levels were related to MACC1 protein levels from immunohistochemistry in FFPE sections; also analyzed for MMR. Chemotherapy-naïve pMMR patients were stratified by MACC1 mRNA and protein expression to establish risk groups based on recurrence-free survival (RFS). Risk stratification from BIOGRID 1 was confirmed in the BIOGRID 2 validation cohort. Pooled BIOGRID datasets produced a best effect-size estimate. RESULTS: In BIOGRID 1, using qRT-PCR and immunohistochemistry for MACC1 detection, pMMR/MACC1-low patients had a lower recurrence probability versus pMMR/MACC1-high patients (5-year RFS of 92% and 67% versus 100% and 68%, respectively). In BIOGRID 2, longer RFS was confirmed for pMMR/MACC1-low versus pMMR/MACC1-high patients (5-year RFS of 100% versus 90%, respectively). In the pooled dataset, 6.5% of patients were pMMR/MACC1-low with no disease recurrence, resulting in a 17% higher 5-year RFS [95% confidence interval (CI) (12.6%-21.3%)] versus pMMR/MACC1-high patients (P = 0.037). Outcomes were similar for pMMR/MACC1-low and deficient MMR (dMMR) patients (5-year RFS of 100% and 96%, respectively). CONCLUSIONS: MACC1 expression stratifies colon cancer patients with unfavorable pMMR status. Stage II colon cancer patients with pMMR/MACC1-low tumors have a similar favorable prognosis to those with dMMR with potential implications for the role of adjuvant therapy.

GPR56 contributes to the development of acute myeloid leukemia in mice.

The G protein-coupled receptor 56 (GPR56) was identified as part of the molecular signature of functionally validated leukemic stem cells isolated from patients with acute myeloid leukemia (AML). This report now demonstrates particularly high expression of GPR56 in patients with mutant NPM1 and FLT3-length mutation and association of high GPR56 expression with inferior prognosis in a large patient cohort treated in two independent multicenter phase III trials. Functional relevance of GPR56 expression was validated in mice, in which co-expression of Gpr56 significantly accelerated HOXA9-induced leukemogenesis and vice versa knockdown of Gpr56 delayed onset of HOXA9/MEIS1-induced AML. Overexpression of Gpr56 grossly changed the molecular phenotype of Hoxa9-transduced cells affecting pathways involved in G protein-coupled receptors (GPRCs) and associated intracellular signaling. Blockage of surface GPR56 by an anti-GPR56 antibody successfully impaired engraftment of primary human AML cells. In summary, these data demonstrate that high expression of GPR56 is able to contribute to AML development and characterize the GPR56 as a potential novel target for antibody-mediated antileukemic strategies.

Symmetrical stabilization of bound Ca2+ ions in a cooperative pair of EF-hands through hydrogen bonding of coordinating water molecules in calbindin D(9k).

Water molecules are found to complete the Ca2+ coordination sphere when a protein fails to provide enough ligating oxygens. Hydrogen bonding of these water molecules to the protein backbone or side chains may contribute favorably to the Ca2+ affinity, as suggested in an earlier study of two calbindin D(9k) mutants [E60D and E60Q; Linse et al. (1994) Biochemistry 33, 12478-12486]. To investigate the generality of this conclusion, another side chain, Gln 22, which hydrogen bonds to a Ca2+-coordinating water molecule in calbindin D(9k), was mutated. Two calbindin D(9k) mutants, (Q22E+P43M) and (Q22N+P43M), were constructed to examine the interaction between Gln 22 and the water molecule in the C-terminal calcium binding site II. Shortening of the side chain, as in (Q22N+P43M), reduces the affinity of binding two calcium ions by a factor of 18 at low ionic strength, whereas introduction of a negative charge, as in (Q22E+P43M), leads to a 12-fold reduction. In 0.15 M KCl, a 7-fold reduction in affinity was observed for both mutants. The cooperativity of Ca2+ binding increases for (Q22E+P43M), while it decreases for (Q22N+P43M). The rates of Ca2+ dissociation are 5.5-fold higher for the double mutants than for P43M at low ionic strength. For both mutants, reduced strength of hydrogen bonding to calcium-coordinating water molecules is a likely explanation for the observed effects on Ca2+ affinity and dissociation. In the apo forms, the (Q22E+P43M) mutant has lower stability toward urea denaturation than (Q22N+P43M) and P43M. 2D (1)H NMR and crystallographic experiments suggest that the structure of (Q22E+P43M) and (Q22N+P43M) is unchanged relative to P43M, except for local perturbations in the loop regions.

Myotonic dystrophy protein kinase phosphorylates the myosin phosphatase targeting subunit and inhibits myosin phosphatase activity.

Myotonic dystrophy protein kinase (DMPK) and Rho-kinase are related. An important function of Rho-kinase is to phosphorylate the myosin-binding subunit of myosin phosphatase (MYPT1) and inhibit phosphatase activity. Experiments were carried out to determine if DMPK could function similarly. MYPT1 was phosphorylated by DMPK. The phosphorylation site(s) was in the C-terminal part of the molecule. DMPK was not inhibited by the Rho-kinase inhibitors, Y-27632 and HA-1077. Several approaches were taken to determine that a major site of phosphorylation was T654. Phosphorylation at T654 inhibited phosphatase activity. Thus both DMPK and Rho-kinase may regulate myosin II phosphorylation.

Identification of the endogenous smooth muscle myosin phosphatase-associated kinase.

Ca(2+) sensitization of smooth muscle contraction involves inhibition of myosin light chain phosphatase (SMPP-1M) and enhanced myosin light chain phosphorylation. Inhibition of SMPP-1M is modulated through phosphorylation of the myosin targeting subunit (MYPT1) by either Rho-associated kinase (ROK) or an unknown SMPP-1M-associated kinase. Activated ROK is predominantly membrane-associated and its putative substrate, SMPP-1M, is mainly myofibrillar-associated. This raises a conundrum about the mechanism of interaction between these enzymes. We present ZIP-like kinase, identified by "mixed-peptide" Edman sequencing after affinity purification, as the previously unidentified SMPP-1M-associated kinase. ZIP-like kinase was shown to associate with MYPT1 and phosphorylate the inhibitory site in intact smooth muscle. Phosphorylation of ZIP-like kinase was associated with an increase in kinase activity during carbachol stimulation, suggesting that the enzyme may be a terminal member of a Ca(2+) sensitizing kinase cascade.

Effect of cyclodextrins on surface and pore properties of soil clay minerals.

Although cyclodextrins are increasingly used in soil decontamination, little is known about their effects on soil physicochemical properties. In this work, the surface and pore properties of randomly methylated beta-cyclodextrin (RAMEB) and three typical clay minerals were characterized, and the effects of RAMEB concentrations on clay minerals were studied using water vapor adsorption-desorption and mercury intrusion porosimetry techniques. As compared to clay minerals, for pure RAMEB very large surface area and volume of nanometer-size pores (micropores) were determined. Energy of interaction with water vapor, volume of micrometer-size pores (mesopores), and fractal dimensions in both pore size ranges of RAMEB were lower than those of the minerals. When increasing amounts of RAMEB were added to the minerals, the surface area and micropore volume decreased and adsorption energy increased. The volume of mesopores decreased after RAMEB treatments for bentonite and kaolin and increased for illite. As deduced from the fractal dimensions increase, the pore structure of the minerals became more complex with RAMEB addition. The observed changes were in general contrary to these expected when RAMEB and minerals coexist as separate, nonreactive phases and suggested strong interaction of RAMEB with clay minerals.

Characterization of the EGF-like module pair 3-4 from vitamin K-dependent protein S using NMR spectroscopy reveals dynamics on three separate time scales and extensive effects from calcium binding.

Protein S, a cofactor of anticoagulant activated protein C, exhibits three high-affinity Ca(2+)-binding sites in a region comprising four EGF modules. The EGF 3-4 module pair constitutes the smallest fragment that retains one high-affinity Ca(2+)-binding site and is therefore useful for investigation of the structural basis of the unusually high-affinity Ca(2+) binding compared to other EGF-containing proteins characterized so far. Extensive chemical shift effects caused by Ca(2+) binding to the EGF 3-4 module pair are observed, particularly from Ca(2+) binding to the high-affinity site in EGF 4. Ca(2+) binding to the high-affinity site in EGF 4 and the low-affinity site in EGF 3 is associated with slow and fast exchange on the NMR time-scale, respectively. We show the presence of two isoforms, characterized by a cis or trans Lys 167-Pro 168 peptide bond, that do not convert on time scales that were accessible to the experiments (k(ex) < 0.2 s(-1)). Both conformers have similar Ca(2+) affinities and backbone dynamics. Further, broadening of (1)H resonances involving residues in the major beta-sheet of EGF 3 and (15)N exchange terms, primarily in the N-terminal part of the protein, indicate the presence of slow exchange on a microsecond to millisecond time scale. (15)N spin relaxation data suggest that the module pair has a well-defined relative orientation between EGF modules 3 and 4 and has a significantly anisotropic rotational diffusion tensor in solution.

1H, 15N and (13)C assignments and secondary structure of the EGF-like module pair 3-4 from vitamin K-dependent protein S.

Vitamin K-dependent protein S, which is a cofactor for activated protein C and thus important for down-regulation of the coagulation cascade, contains several Ca(2+)-binding sites with unusually high affinity. The 89 amino acid fragment constituting the third and fourth epidermal growth factor-like (EGF) modules of protein S is the smallest fragment that retains high-affinity Ca(2+) binding and is therefore useful for investigating the structural basis of this property. Heteronuclear multidimensional nuclear magnetic resonance experiments were used to obtain extensive assignments of the (1)H, 15N and (13)C resonances of the module pair with one Ca(2+) bound in EGF 4. In addition, nearly complete assignments of the (1)H resonances of the isolated Ca(2+)-free EGF 3 module were obtained. The assignment process was complicated by broadening of several resonances, spectral heterogeneity caused by cis-trans isomerisation of the peptide bond preceding Pro-168, and dimerisation. Analysis of weighted average secondary chemical shifts, (3)J(HNHalpha) coupling constants, and NOE connectivities suggest that both EGF modules in this fragment adhere to the classical secondary structure of EGF modules, consisting of one major and one minor anti-parallel beta-sheet.

Calcium-binding EGF-like modules in coagulation proteinases: function of the calcium ion in module interactions.

Epidermal growth factor (EGF)-like modules are involved in protein-protein interactions and are found in numerous extracellular proteins and membrane proteins. Among these proteins are enzymes involved in blood coagulation, fibrinolysis and the complement system as well as matrix proteins and cell surface receptors such as the EGF precursor, the low density lipoprotein receptor and the developmentally important receptor, Notch. The coagulation enzymes, factors VII, IX and X and protein C, all have two EGF-like modules, whereas the cofactor of activated protein C, protein S, has four EGF-like modules in tandem. Certain of the cell surface receptors have numerous EGF modules in tandem. A subset of EGF modules bind one Ca(2+). The Ca(2+)-binding sequence motif is coupled to a sequence motif that brings about beta-hydroxylation of a particular Asp/Asn residue. Ca(2+)-binding to an EGF module is important to orient neighboring modules relative to each other in a manner that is required for biological activity. The Ca(2+) affinity of an EGF module is often influenced by its N-terminal neighbor, be it another EGF module or a module of another type. This can result in an increase in Ca(2+) affinity of several orders of magnitude. Point mutations in EGF modules that involve amino acids which are Ca(2+) ligands result in the biosynthesis of biologically inactive proteins. Such mutations have been identified, for instance, in factor IX, causing hemophilia B, in fibrillin, causing Marfan syndrome, and in the low density lipoprotein receptor, causing hypercholesterolemia. In this review the emphasis will be on the coagulation factors.

EGF-like module pair 3-4 in vitamin K-dependent protein S: modulation of calcium affinity of module 4 by module 3, and interaction with factor X.

Calcium-binding epidermal growth factor (EGF)-like modules are found in numerous extracellular and membrane proteins involved in such diverse processes as blood coagulation, lipoprotein metabolism, determination of cell fate, and cell adhesion. Vitamin K-dependent protein S, a cofactor of the anticoagulant enzyme activated protein C, has four EGF-like modules in tandem with the three C-terminal modules each harbouring a Ca(2+)-binding consensus sequence. Recombinant fragments containing EGF modules 1-4 and 2-4 have two Ca(2+)-binding sites with dissociation constants ranging from 10(-8) to 10(-5) M. Module-module interactions that greatly influence the Ca(2+) affinity of individual modules have been identified. As a step towards an analysis of the structural basis of the high Ca(2+) affinity, we expressed the Ca(2+)-binding EGF pair 3-4 from human protein S. Correct folding was shown by (1)H NMR spectroscopy. Calcium-binding properties of the C-terminal module were determined by titration with chromophoric chelators; binding to the low-affinity N-terminal site was monitored by (1)H-(15)N NMR spectroscopy. At physiological pH and ionic strength, the dissociation constants for Ca(2+) binding were 1.0x10(-6) M and 4. 8x10(-3) M for modules 4 and 3, respectively, i.e. the calcium affinity of the C-terminal site was about 5000-fold higher than that of the N-terminal site. Moreover, the Ca(2+) affinity of EGF 4, in the pair 3-4, was about 9000-fold higher than that of synthetic EGF 4. The EGF modules in protein S are known to mediate the interaction with factor Xa. We have now found modules 3-4 to be involved in this interaction. However, the individual modules 3 and 4 manifested no measurable activity.

Protein phosphatase 2A plays a role in the suckling-induced changes in the responsiveness of pituitary mammotropes.

It is well established that PRL secretion is under a tonic inhibition exercised by the hypothalamic dopamine (DA). One feature of this regulation is an immediate withdrawal reaction (elevation of PRL release) of mammotropes after disruption of hypothalamic influence. Although plasma PRL rises rapidly, the suckling stimulus does not cause an acute diminution of hypothalamic DA, but, as we have previously demonstrated, it results in an almost immediate (within 10 min) desensitization of mammotropes as indicated by the change in dose response of DA to inhibit PRL release. Our present investigations relate to the phenomenon of this change in responsiveness of PRL cells. This was accomplished by using the reverse hemolytic plaque assay to evaluate the secretory characteristics of individual PRL secretors derived from lactating rats either before or after a 10-min suckling stimulus. To investigate the mechanism of these changes, the binding characteristics of [3H]spiperone on pituitary membranes from nonsuckled and suckled rats have been compared, and the possible involvement of dephosphorylating enzymes was tested by using okadaic acid (OA) in a dose of 2 nM that preferentially and selectively inhibits protein phosphatase-2A (PP2A) activity. We have also determined the activities of PP1 and PP2A in pituitary tissue samples as well as in enzymatically dispersed cells. Mammotropes from nonsuckled rats exhibited a depression of PRL release after both DA and OA treatment and an elevation after withdrawal of DA. This suggests that the secretory response of mammotropes obtained from nonsuckled rats still shows those two responses that are characteristic of the tonic inhibitory regulation. In contrast, superimposition of suckling in vivo or application of OA together with DA pretreatment in cells from nonsuckled rats in vitro resulted in a disappearance of the dissociation-induced elevation of PRL release, indicating an abolishment of the tonic inhibitory action of DA. Evidence is also presented that the PP2A, but not the PP1, activity of the anterior lobe is significantly lower after a 10-min suckling stimulus. Moreover, DA is able to decrease PP2A activity in dispersed pituitary cells obtained from nonsuckled, but not from suckled, animals. In contrast, there were no differences in either the affinity or the number of binding sites between nonsuckled and suckled rats. Taken together, our results suggest that the suckling-induced decrease in PP2A activity plays a role in the uncoupling of D2 receptors on mammotropes from the tonic inhibitory signaling pathway.

Solution structure of the N-terminal EGF-like domain from human factor VII.

Blood coagulation is initiated by Ca(2+)-dependent binding of coagulation factor VIIa (FVIIa) to its cofactor, tissue factor (TF). The TF:FVIIa complex activates factors IX and X, ultimately leading to the formation of thrombin and the coagulation of blood. FVII consists of an N-terminal gamma-carboxyglutamic-acid-containing (Gla) domain followed by two epidermal growth factor (EGF) like domains, the first of which can bind one Ca2+ ion (Kd approximately 150 microM) and a C-terminal serine protease domain. Using 1H nuclear magnetic resonance spectroscopy, we have determined the solution structure of a synthetic N-terminal EGF-like domain (EGF1) of human FVII (residues 45-85) in the absence of Ca2+. A comparison of this structure of apo EGF1 with the Ca(2+)-bound EGF1 in the complex of FVIIa and TF [Banner, D. W., et al. (1996) Nature 380, 41-46] suggests that the structural changes in the EGF1 domain upon Ca2+ binding are minor and are concentrated near the Ca(2+)-binding site, which is facing away from the TF interaction surface. Amino acid side chains that are crucial for the binding of FVII to TF show a similar conformation in both structures and are therefore unlikely to directly influence the Ca(2+)-dependent binding of FVII to TF. As Ca2+ binding to EGF1 does not lead to a conformational change in the residues constituting the interaction surface for binding to TF, our results are consistent with the idea that the altered orientation between the Gla and EGF1 domains that result from Ca2+ binding is responsible for the increased affinity of FVII/FVIIa for TF.

Identification and localization of myosin phosphatase in human platelets.

Type 1 (PP1) and type 2A (PP2A) phosphatase activity was measured in three subcellular fractions of human platelets. About 80% of the activity was in the high-speed supernatant. Western blots showed that the catalytic subunit of PP1 (PP1c), including alpha- and delta-isoforms, was present in each fraction, but the level of the catalytic subunit of PP2A was very low in the low-speed pellet (cytoskeletal fraction). Various antibodies detected a subunit similar to the 130 kDa subunit (M130) of myosin phosphatase (MP) of smooth muscle in the low- and the high-speed pellets of human platelets. PP1c and associated proteins were isolated by microcystin-Sepharose. Many proteins were separated from each fraction, including myosin, actin and PP1c. M130 was separated only from the low-speed and the high-speed pellets. Kinase activities were detected in the unbound fractions, and fractions from the low- and high-speed pellets phosphorylated M130 and myosin respectively. Treatment of platelets with calyculin A increased the phosphorylation level of many proteins, including myosin heavy- and light-chains, and caused association of cytoskeletal proteins with the low-speed pellet. No marked change in the distribution of PP1c and M130 was detected. These results suggest that the MP in human platelets is composed of PP1c plus a subunit similar to M130 of the smooth muscle phosphatase.

The possible role of protein phosphatase 2A in the sodium sensitivity of the receptor binding of opiate antagonists naloxone and naltrindole.

In striatal membrane preparation used for receptor binding experiments high levels of protein phosphatase 1 and 2A activities were detected using [32P]phosphorylase a as substrate. Sodium chloride decreased the activity of protein phosphatase 2A and increased the activity of protein phosphatase 1 in a concentration-dependent manner. Sodium chloride facilitated the saturation binding of naloxone and naltrindole in rat striatal membrane preparation preincubated with ATP (50 microM) and MgCl2 (5 mM). Preincubation with calyculin A (1 nM) further increased the binding of naloxone. Addition of okadaic acid in a concentration of 2 nM, which is specific for the inhibition of protein phosphatase 2A, augmented the number of binding sites of naloxone or naltrindole. The results suggest a protein phosphatase-dependent regulation of the binding of opiate ligands in the striatum.

Quantitation of protein phosphatase 1 and 2A in extracts of the budding yeast and fission yeast.

Serine/threonine protein phosphatases are also involved in the control of cell division. The aim of the present study was to compare the activity of protein phosphatase 1 (PP1) and 2A (PP2A) in cell extracts of the budding and fission yeast, made at different phases of growth. The activities of PP1 and PP2A toward phosphorylase were similar in extracts of S. cerevisiae. In S. pombe extracts, PP1 was responsible for more than 80% of the phosphorylase phosphatase activity. Ammonium sulfate-ethanol treatment increased the specific activity of the phosphatases and the percentage of PP2A in S. cerevisiae extracts. No increase in the proportion of PP2A was observed upon the same treatment of S. pombe extracts. The above results were confirmed by fractionation of PP1 and PP2A activities on a heparin-Sepharose column. The proportion of PP1 and PP2A activities did not change significantly during exponential cell growth but cells from stationary phase exhibited lower phosphatase activities. These results may indicate a lower level of expression of the PP2A genes in S. pombe and/or differences in the structure of the holoenzymes or their regulators in the two genera.

Purification and characterization of three distinct types of protein phosphatase catalytic subunits in bovine platelets.

The catalytic subunits of bovine platelet protein phosphatases were separated into three distinct forms by chromatography on heparin-Sepharose. Each phosphatase was further purified to apparent homogeneity as judged in sodium dodecyl sulfate-polyacrylamide gel yielding single protein bands of 37, 41, and 36 kDa. The 37-kDa phosphatase was excluded from heparin-Sepharose and preferentially dephosphorylated the alpha-subunit of phosphorylase kinase. It was stimulated by polycations (polybrene or histone H1) and was inhibited by okadaic acid (IC50 = 0.3 nM), but its activity was not influenced by inhibitor-2 or heparin. The 41-kDa phosphatase was eluted from heparin-Sepharose by 0.20-0.25 M NaCl and preferentially dephosphorylated the beta-subunit of phosphorylase kinase. It was stimulated by polycations and inhibited by okadaic acid (IC50 = 2 nM), but its activity was not affected by inhibitor-2 or heparin. The 36-kDa phosphatase was eluted from heparin-Sepharose by 0.45-0.50 M NaCl and preferentially dephosphorylated the beta-subunit of phosphorylase kinase. It was inhibited by inhibitor-2, heparin, histone H1, and okadaic acid (IC50 = 70 nM). The 37- and 36-kDa phosphatases can be classified as type-2A and type-1 enzymes, respectively. The 41-kDa phosphatase does not precisely fit the criteria of either type, showing only partial similarities to both type-1 and type-2A enzymes and it may represent a novel type of protein phosphatase in bovine platelets.