Biochemical characterization and deletion analysis of recombinant human protein phosphatase 2Cα

The use of protein phosphatase inhibitors has been instrumental in defining the intracellular roles of protein phosphatase 1 (PP1), PP2A and PP2B. Identification of the role of PP2C in vivo has been hampered, in part, by the unavailability of specific inhibitors. In order to facilitate the identification of novel and specific inhibitors of PP2C by random screening of compounds, and to further characterize this enzyme at the molecular level by site-directed mutagenesis and X-ray crystallography, we have expressed active recombinant human PP2Cα (rPP2Cα) in Escherichia coli. Biochemical characterization of rPP2Cα showed that it could hydrolyse p-nitrophenyl phosphate (pNPP) although, in contrast with native PP2C, this was not stimulated by Mg2+. As with native PP2C, okadaic acid failed to inhibit rPP2Cα, whereas 50 mM NaF dramatically inhibited its activity. An alignment of the amino acid sequence of AMP-activated protein kinase (AMPK) with those of other serine/threonine protein kinases around the regulatory phosphorylation site (subdomains VII–VIII) revealed a high degree of conservation. Phosphopeptides derived from this region of AMPK and containing the almost invariant threonine (Thr172 in AMPK) were found to be good substrates for rPP2Cα. We also showed that rPP2Cα can inactivate AMPK, but only in the presence of Mg2+. To define the regions of PP2Cα important for catalytic activity, we expressed a number of truncated proteins based on the sequence and proposed domain structure of the PP2Cα homologue from Paramecium tetraurelia. Deletion of 75 residues (9 kDa) from the C-terminus appeared to have little effect on the catalytic activity using pNPP, phosphopeptides or AMPK as substrates. This suggests that the residues important in catalysis lie elsewhere in the protein. A further deletion of the C-terminus led to a completely inactive and very poorly soluble protein.

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A kinase cascade on the yeast lysosomal vacuole regulates its membrane dynamics: conserved kinase Env7 is phosphorylated by casein kinase Yck3

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.012346 ◽

2020 ◽

Vol 295 (34) ◽

pp. 12262-12278

Author(s):

Surya P. Manandhar ◽

Ikha M. Siddiqah ◽

Stephanie M. Cocca ◽

Editte Gharakhanian

Keyword(s):

Membrane Fusion ◽

Phosphorylation Site ◽

Casein Kinase ◽

Membrane Dynamics ◽

Vacuolar Membrane ◽

Site Directed Mutagenesis ◽

Casein Kinase I ◽

C Terminus ◽

Kinase Cascade

Membrane fusion/fission is a highly dynamic and conserved process that responds to intra- and extracellular signals. Whereas the molecular machineries involved in membrane fusion/fission have been dissected, regulation of membrane dynamics remains poorly understood. The lysosomal vacuole of budding yeast (Saccharomyces cerevisiae) has served as a seminal model in studies of membrane dynamics. We have previously established that yeast ENV7 encodes an ortholog of STK16-related kinases that localizes to the vacuolar membrane and downregulates vacuolar membrane fusion. Additionally, we have previously reported that Env7 phosphorylation in vivo depends on YCK3, a gene that encodes a vacuolar membrane casein kinase I (CKI) homolog that nonredundantly functions in fusion regulation. Here, we report that Env7 physically interacts with and is directly phosphorylated by Yck3. We also establish that Env7 vacuole fusion/fission regulation and vacuolar localization are mediated through its Yck3-dependent phosphorylation. Through extensive site-directed mutagenesis, we map phosphorylation to the Env7 C terminus and confirm that Ser-331 is a primary and preferred phosphorylation site. Phospho-deficient Env7 mutants were defective in negative regulation of membrane fusion, increasing the number of prominent vacuoles, whereas a phosphomimetic substitution at Ser-331 increased the number of fragmented vacuoles. Bioinformatics approaches confirmed that Env7 Ser-331 is within a motif that is highly conserved in STK16-related kinases and that it also anchors an SXXS CKI phosphorylation motif (328SRFS331). This study represents the first report on the regulatory mechanism of an STK16-related kinase. It also points to regulation of vacuolar membrane dynamics via a novel Yck3–Env7 kinase cascade.

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Saccharomyces cerevisiae ubiquitin-like protein Rub1 conjugates to cullin proteins Rtt101 and Cul3 in vivo

Biochemical Journal ◽

10.1042/bj20030755 ◽

2004 ◽

Vol 377 (2) ◽

pp. 459-467 ◽

Cited By ~ 17

Author(s):

Jose M. LAPLAZA ◽

Magnolia BOSTICK ◽

Derek T. SCHOLES ◽

M. Joan CURCIO ◽

Judy CALLIS

Keyword(s):

Saccharomyces Cerevisiae ◽

Protein A ◽

Fold Increase ◽

Lysine Residue ◽

Reading Frame ◽

E3 Ligases ◽

C Terminus ◽

Dependent Modification ◽

F Box Protein

In Saccharomyces cerevisiae, the ubiquitin-like protein Rub1p (related to ubiquitin 1 protein) covalently attaches to the cullin protein Cdc53p (cell division cycle 53 protein), a subunit of a class of ubiquitin E3 ligases named SCF (Skp1–Cdc53–F-box protein) complex. We identified Rtt101p (regulator of Ty transposition 101 protein, where Ty stands for transposon of yeast), initially found during a screen for proteins to confer retrotransposition suppression, and Cul3p (cullin 3 protein), a protein encoded by the previously uncharacterized open reading frame YGR003w, as two new in vivo targets for Rub1p conjugation. These proteins show significant identity with Cdc53p and, therefore, are cullin proteins. Modification of Cul3p is eliminated by deletion of the Rub1p pathway through disruption of either RUB1 or its activating enzyme ENR2/ULA1. The same disruptions in the Rub pathway decreased the percentage of total Rtt101p that is modified from approx. 60 to 30%. This suggests that Rtt101p has an additional RUB1- and ENR2-independent modification. All modified forms of Rtt101p and Cul3p were lost when a single lysine residue in a conserved region near the C-terminus was replaced by an arginine residue. These results suggest that this lysine residue is the site of Rub1p-dependent and -independent modifications in Rtt101p and of Rub1p-dependent modification in Cul3p. An rtt101Δ strain was hypersensitive to thiabendazole, isopropyl (N-3-chlorophenyl) carbamate and methyl methanesulphonate, but rub1Δ strains were not. Whereas rtt101Δ strains exhibited a 14-fold increase in Ty1 transposition, isogenic rub1Δ strains did not show statistically significant increases. Rtt101K791Rp, which cannot be modified, complemented for Rtt101p function in a transposition assay. Altogether, these results suggest that neither the RUB1-dependent nor the RUB1-independent form of Rtt101p is required for Rtt101p function. The identification of additional Rub1p targets in S. cerevisiae suggests an expanded role for Rub in this organism.

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Negative regulation of Raf-1 by phosphorylation of serine 621.

Molecular and Cellular Biology ◽

10.1128/mcb.16.10.5409 ◽

1996 ◽

Vol 16 (10) ◽

pp. 5409-5418 ◽

Cited By ~ 151

Author(s):

H Mischak ◽

T Seitz ◽

P Janosch ◽

M Eulitz ◽

H Steen ◽

...

Keyword(s):

Catalytic Activity ◽

Kinase Activity ◽

Phosphorylation Site ◽

Kinase Domain ◽

Negative Regulation ◽

Catalytic Function ◽

Dependent Protein ◽

The One

The elevation of cyclic AMP (cAMP) levels in the cell downregulates the activity of the Raf-1 kinase. It has been suggested that this effect is due to the activation of cAMP-dependent protein kinase (PKA), which can directly phosphorylate Raf-1 in vitro. In this study, we confirmed this hypothesis by coexpressing Raf-1 with the constitutively active catalytic subunit of PKA, which could fully reproduce the inhibition previously achieved by cAMP. PKA-phosphorylated Raf-1 exhibits a reduced affinity for GTP-loaded Ras as well as impaired catalytic activity. As the binding to GTP-loaded Ras induces Raf-1 activation in the cell, we examined which mechanism is required for PKA-mediated Raf-1 inhibition in vivo. A Raf-1 point mutant (RafR89L), which is unable to bind Ras, as well as the isolated Raf-1 kinase domain were still fully susceptible to inhibition by PKA, demonstrating that the phosphorylation of the Raf-1 kinase suffices for inhibition. By the use of mass spectroscopy and point mutants, PKA phosphorylation site was mapped to a single site in the Raf-1 kinase domain, serine 621. Replacement of serine 621 by alanine or cysteine or destruction of the PKA consensus motif by changing arginine 618 resulted in the loss of catalytic activity. Notably, a mutation of serine 619 to alanine did not significantly affect kinase activity or regulation by activators or PKA. Changing serine 621 to aspartic acid yielded a Raf-1 protein which, when expressed to high levels in Sf-9 insect cells, retained a very low inducible kinase activity that was resistant to PKA downregulation. The purified Raf-1 kinase domain displayed slow autophosphorylation of serine 621, which correlated with a decrease in catalytic function. The Raf-1 kinase domain activated by tyrosine phosphorylation could be downregulated by PKA. Specific removal of the phosphate residue at serine 621 reactivated the catalytic activity. These results are most consistent with a dual role of serine 621. On the one hand, serine 621 appears essential for catalytic activity; on the other hand, it serves as a phosphorylation site which confers negative regulation.

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Regulation of SulA cleavage by Lon protease by the C-terminal amino acid of SulA, histidine

Biochemical Journal ◽

10.1042/bj3580473 ◽

2001 ◽

Vol 358 (2) ◽

pp. 473-480 ◽

Cited By ~ 14

Author(s):

Yoshiyuki ISHII ◽

Fumio AMANO

Keyword(s):

Amino Acid ◽

Protein A ◽

Lon Protease ◽

Histidine Residue ◽

High Accumulation ◽

C Terminus ◽

A Cell ◽

Cell Division Inhibitor

SulA protein, a cell division inhibitor in Escherichia coli, is degraded by Lon protease. The C-terminal eight residues of SulA have been shown to be recognized by Lon; however, it remains to be elucidated which amino acid in the C-terminus of SulA is critical for the recognition of SulA by Lon. To clarify this point, we constructed mutants of SulA with changes in the C-terminal residues, and examined the accumulation and stability of the resulting mutant SulA proteins in vivo. Substitution of the extreme C-terminal histidine residue with another amino acid led to marked accumulation and high stability of SulA in lon+ cells. A SulA mutant in which the C-terminal eight residues were deleted (SulAC161) showed high accumulation and stability, but the addition of histidine to the C-terminus of SulAC161 (SulAC161+H) made it labile. Similarly, SulAC161+H fused to maltose-binding protein (MBP–SulAC161+H) formed a tight complex with and was degraded rapidly by Lon in vitro. Histidine competitively inhibited the degradation of MBP–SulA by Lon, while other amino acids did not. These results suggest that the histidine residue at the extreme C-terminus of SulA is recognized specifically by Lon, leading to a high-affinity interaction between SulA and Lon.

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Mechanisms of Apoptosis Induction by BH3 Inhibitor ABT-737 in AML.

Blood ◽

10.1182/blood.v106.11.244.244 ◽

2005 ◽

Vol 106 (11) ◽

pp. 244-244

Author(s):

Michael Andreeff ◽

Rooha Contractor ◽

Peter P. Ruvolo ◽

Xingming Deng ◽

Ismael Samudio ◽

...

Keyword(s):

Cell Lines ◽

Phosphorylation Site ◽

Leukemia Cell ◽

Mek Inhibitor ◽

Chemotherapeutic Agents ◽

Site Directed Mutagenesis ◽

Apoptotic Pathway ◽

Induced Apoptosis ◽

In Cells

Abstract Bcl2 family proteins are key regulators of apoptosis. Aberrations in Bcl2 levels are known to promote tumorigenesis and chemoresistance. Thus, strategies to target Bcl2 will likely provide effective therapies for malignancies such as acute myeloid leukemia (AML). In this report, we investigate mechanisms of action of the novel small molecule Bcl2 inhibitor ABT-737 in AML. ABT-737 effectively killed AML patient blast cells and colony-forming cell lines at nanomolar concentrations with no effect on normal hematopoietic cells. Notably, CD34+38−123+ AML stem cells are highly sensitive to the compound. ABT-737-induced apoptosis is initiated by disruption of Bcl2:Bax dimers and activation of the intrinsic apoptotic pathway. ABT-737 works synergistically with chemotherapeutic agents such as ara-C and doxorubicin. To investigate the role of Bcl-2 phosphorylation in the sensitivity to BH3 inhibitor, we used IL-3 dependent NSF.N1/H7 mouse myeloid cells modified by site-directed mutagenesis to produce various Bcl-2 phospho-mutants. NSF.N1/H7 cells stably transfected with phosphomimetic T69E/S70E/S87E (EEE) Bcl-2 mutants were resistant to ABT-737 (IC50>500 nM) as compared to cells expressing wt-Bcl-2 or the nonphosphorylatable T69A/S70A/S87A (AAA) Bcl2 mutants (IC50s of 50 and 25 nM). Consistent with a mechanism whereby increased Bcl2 phosphorylation impedes ABT-737 suppression of Bcl2 dimerization with Bax, ABT-737 potently blocked Bcl2:Bax association in cells expressing exogenous WT Bcl2 and AAA mutant Bcl2 but not in cells expressing exogenous phosphomimetic EEE mutant Bcl2. Since the S70E phosphorylation site of Bcl-2 is a known ERK substrate, we examined combined effects of ABT-737 and MEK inhibitor PD98059 in OCI-AML3 cells resistant to ABT-737 alone. The combined activity of PD98059 and ABT-737, evaluated by isobologram analysis, revealed a striking synergistic interaction between the MEK and BH3 inhibitors, with combination indices (CI) of 0.08±0.003. OCI-AML3 cells exhibit the highest expression of Mcl-1 among the acute leukemia cell lines tested. We propose that loss of Mcl-1 expression as a result of suppression of ERK may also be involved in the ability of PD98059 to enhance ABT-737-induced apoptosis. siRNA to Mcl-1 strikingly sensitized OCI-AML3 cells to ABT-induced apoptosis (14% apoptosis in parental cells at 2.5μM ABT-737, 64% apoptosis in siRNA-transfected cells at 10-fold lower concentration of 0.25μM). We have further demonstrated that ABT-737 reduced leukemia burden and significantly (p=0.0018) prolonged survival of mice in an in vivo mouse model. These findings suggest that: 1) ABT-737 reduces apoptosis through disruption of Bcl2:Bax heterodimers; 2) its activity is limited by Bcl2 phosphorylation and Mcl-1 overexpression; 3) combination with MEK inhibition results in inhibition of Bcl2 phosphorylation, downregulation of Mcl-1 and dramatic enhancement of ABT-737-induced apoptosis in AML.

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A High-affinity Interaction with ADP-Actin Monomers Underlies the Mechanism and In Vivo Function of Srv2/cyclase-associated Protein

Molecular Biology of the Cell ◽

10.1091/mbc.e04-06-0444 ◽

2004 ◽

Vol 15 (11) ◽

pp. 5158-5171 ◽

Cited By ~ 74

Author(s):

Pieta K. Mattila ◽

Omar Quintero-Monzon ◽

Jamie Kugler ◽

James B. Moseley ◽

Steven C. Almo ◽

...

Keyword(s):

Site Directed Mutagenesis ◽

Actin Binding ◽

Actin Monomer ◽

Nucleotide Exchange ◽

Actin Organization ◽

C Terminus ◽

Actin Binding Domain ◽

Affinity Interaction

Cyclase-associated protein (CAP), also called Srv2 in Saccharomyces cerevisiae, is a conserved actin monomer-binding protein that promotes cofilin-dependent actin turnover in vitro and in vivo. However, little is known about the mechanism underlying this function. Here, we show that S. cerevisiae CAP binds with strong preference to ADP-G-actin (Kd 0.02 μM) compared with ATP-G-actin (Kd 1.9 μM) and competes directly with cofilin for binding ADP-G-actin. Further, CAP blocks actin monomer addition specifically to barbed ends of filaments, in contrast to profilin, which blocks monomer addition to pointed ends of filaments. The actin-binding domain of CAP is more extensive than previously suggested and includes a recently solved β-sheet structure in the C-terminus of CAP and adjacent sequences. Using site-directed mutagenesis, we define evolutionarily conserved residues that mediate binding to ADP-G-actin and demonstrate that these activities are required for CAP function in vivo in directing actin organization and polarized cell growth. Together, our data suggest that in vivo CAP competes with cofilin for binding ADP-actin monomers, allows rapid nucleotide exchange to occur on actin, and then because of its 100-fold weaker binding affinity for ATP-actin compared with ADP-actin, allows other cellular factors such as profilin to take the handoff of ATP-actin and facilitate barbed end assembly.

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Identification of Residues Important for the Activity ofHaloferax volcaniiAglD, a Component of the Archaeal N-Glycosylation Pathway

Archaea ◽

10.1155/2010/315108 ◽

2010 ◽

Vol 2010 ◽

pp. 1-9 ◽

Cited By ~ 7

Author(s):

Lina Kaminski ◽

Jerry Eichler

Keyword(s):

Biochemical Characterization ◽

Haloferax Volcanii ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Amino Acid Residues ◽

Natural Substrate ◽

Identify Amino Acid ◽

Glycosylation Pathway

InHaloferax volcanii, AglD adds the final hexose to the N-linked pentasaccharide decorating the S-layer glycoprotein. Not knowing the natural substrate of the glycosyltransferase, together with the challenge of designing assays compatible with hypersalinity, has frustrated efforts at biochemical characterization of AglD activity. To circumvent these obstacles, an in vivo assay designed to identify amino acid residues important for AglD activity is described. In the assay, restoration of AglD function in anHfx. volcanii aglDdeletion strain transformed to express plasmid-encoded versions of AglD, generated through site-directed mutagenesis at positions encoding residues conserved in archaeal homologues of AglD, is reflected in the behavior of a readily detectable reporter of N-glycosylation. As such Asp110 and Asp112 were designated as elements of the DXD motif of AglD, a motif that interacts with metal cations associated with nucleotide-activated sugar donors, while Asp201 was predicted to be the catalytic base of the enzyme.

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Human xylosyltransferase I: functional and biochemical characterization of cysteine residues required for enzymic activity

Biochemical Journal ◽

10.1042/bj20041206 ◽

2005 ◽

Vol 386 (2) ◽

pp. 227-236 ◽

Cited By ~ 15

Author(s):

Sandra MÜLLER ◽

Manuela SCHÖTTLER ◽

Sylvia SCHÖN ◽

Christian PRANTE ◽

Thomas BRINKMANN ◽

...

Keyword(s):

Catalytic Activity ◽

Biochemical Characterization ◽

Active Form ◽

Inhibitory Effect ◽

Enzymic Activity ◽

Site Directed Mutagenesis ◽

Cysteine Residues ◽

Thiol Groups ◽

Wild Type ◽

Free Thiol

XT-I (xylosyltransferase I) is the initial enzyme in the post-translational biosynthesis of glycosaminoglycan chains in proteoglycans. To gain insight into the structure–function relationship of the enzyme, a soluble active form of human XT-I was expressed in High Five insect cells with an apparent molecular mass of 90 kDa. Analysis of the electrophoretic mobility of the protein under non-reducing and reducing conditions indicated that soluble XT-I does not form homodimers through disulphide bridges. In addition, the role of the cysteine residues was investigated by site-directed mutagenesis combined with chemical modifications of XT-I by N-phenylmaleimide. Replacement of Cys471 or Cys574 with alanine led to a complete loss of catalytic activity, indicating the necessity of these residues for maintaining an active conformation of soluble recombinant XT-I by forming disulphide bonds. On the other hand, N-phenylmaleimide treatment showed no effect on wild-type XT-I but strongly inactivated the cysteine mutants in a dose-dependant manner, indicating that seven intramolecular disulphide bridges are formed in wild-type XT-I. The inhibitory effect of UDP on the XT-I activity of C561A (Cys561→Ala) mutant enzyme was significantly reduced compared with all other tested cysteine mutants. In addition, we tested for binding to UDP-agarose beads. The inactive mutants revealed no significantly different nucleotide-binding properties. Our study demonstrates that recombinant XT-I is organized as a monomer with no free thiol groups and strongly suggests that the catalytic activity does not depend on the presence of free thiol groups, furthermore, we identified five cysteine residues which are critical for enzyme activity.

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Expression of a myosin regulatory light chain phosphorylation site mutant complements the cytokinesis and developmental defects of Dictyostelium RMLC null cells.

The Journal of Cell Biology ◽

10.1083/jcb.127.6.1945 ◽

1994 ◽

Vol 127 (6) ◽

pp. 1945-1955 ◽

Cited By ~ 52

Author(s):

B D Ostrow ◽

P Chen ◽

R L Chisholm

Keyword(s):

Atpase Activity ◽

Light Chain ◽

Contractile Activity ◽

Phosphorylation Site ◽

Regulatory Light Chain ◽

Site Directed Mutagenesis ◽

Cellular Functions ◽

Developmental Defects

In a number of systems phosphorylation of the regulatory light chain (RMLC) of myosin regulates the activity of myosin. In smooth muscle and vertebrate nonmuscle systems RMLC phosphorylation is required for contractile activity. In Dictyostelium discoideum phosphorylation of the RMLC regulates both ATPase activity and motor function. We have determined the site of phosphorylation on the Dictyostelium RMLC and used site-directed mutagenesis to replace the phosphorylated serine with an alanine. The mutant light chain was then expressed in RMLC null Dictyostelium cells (mLCR-) from an actin promoter on an integrating vector. The mutant RMLC was expressed at high levels and associated with the myosin heavy chain. RMLC bearing a ser13ala substitution was not phosphorylated in vitro by purified myosin light chain kinase, nor could phosphate be detected on the mutant RMLC in vivo. The mutant myosin had reduced actin-activated ATPase activity, comparable to fully dephosphorylated myosin. Unexpectedly, expression of the mutant RMLC rescued the primary phenotypic defects of the mlcR- cells to the same extent as did expression of wild-type RMLC. These results suggest that while phosphorylation of the Dictyostelium RMLC appears to be tightly regulated in vivo, it is not essential for myosin-dependent cellular functions.

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Regulation of chromosome segregation by Glc8p, a structural homolog of mammalian inhibitor 2 that functions as both an activator and an inhibitor of yeast protein phosphatase 1.

Molecular and Cellular Biology ◽

10.1128/mcb.15.11.6064 ◽

1995 ◽

Vol 15 (11) ◽

pp. 6064-6074 ◽

Cited By ~ 66

Author(s):

H Y Tung ◽

W Wang ◽

C S Chan

Keyword(s):

Protein Kinase ◽

Chromosome Segregation ◽

Protein Phosphatase ◽

Site Directed Mutagenesis ◽

Protein Phosphatase 1 ◽

Temperature Sensitive ◽

Loss Of Function ◽

Yeast Saccharomyces Cerevisiae ◽

Growth Phenotype

The Ipl1 protein kinase is essential for proper chromosome segregation and cell viability in the budding yeast Saccharomyces cerevisiae. We have previously shown that the temperature-sensitive growth phenotype of conditional ipl1-1ts mutants can be suppressed by a partial loss-of-function mutation in the GLC7 gene, which encodes the catalytic subunit (PP1C) of protein phosphatase 1, thus suggesting that this enzyme acts in opposition to the Ipl1 protein kinase in regulating yeast chromosome segregation. We report here that the Glc8 protein, which is related in primary sequence to mammalian inhibitor 2, also participates in this regulation. Like inhibitor 2, the Glc8 protein is heat stable, exhibits anomalous electrophoretic mobility, and functions in vitro as an inhibitor of yeast as well as rabbit skeletal muscle PP1C. Interestingly, overexpression as well as deletion of the GLC8 gene results in a partial suppression of the temperature-sensitive growth phenotype of ipl1ts mutants and also moderately reduces the amount of protein phosphatase 1 activity which is assayable in crude yeast lysates. In addition, the chromosome missegregation phenotype caused by an increase in the dosage of GLC7 is totally suppressed by the glc8-delta 101::LEU2 deletion mutation. These findings together suggest that the Glc8 protein is involved in vivo in the activation of PP1C and that when the Glc8 protein is overproduced, it may also inhibit PP1C function. Furthermore, site-directed mutagenesis studies of GLC8 suggest that Thr-118 of the Glc8 protein, which is equivalent to Thr-72 of inhibitor 2, may play a central role in the ability of this protein to activate and/or inhibit PP1C in vivo.

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