Viktor Dombrádi

Phosphorylation blocks the activity of tubulin polymerization promoting protein (TPPP): Identification of sites targeted by different kinases

Tubulin polymerization-promoting protein (TPPP), an unfolded brain-specific protein interacts with the tubulin/microtubule system in vitro and in vivo, and is enriched in human pathological brain inclusions. Here we show that TPPP induces tubulin self-assembly into intact frequently bundled microtubules, and that the phosphorylation of specific sites distinctly affects the function of TPPP. In vitro phosphorylation of wild type and the truncated form (Δ3-43TPPP) of human recombinant TPPP was performed by kinases involved in brain-specific processes. A stoichiometry of 2.9 ± 0.3, 2.2 ± 0.3, and 0.9 ± 0.1 mol P/mol protein with ERK2, cyclin-dependent kinase 5 (Cdk5), and cAMP-dependent protein kinase (PKA), respectively, was revealed for the full-length protein, and 0.4-0.5 mol P/mol protein was detected with all three kinases when the N-terminal tail was deleted. The phosphorylation sites Thr14, Ser18, Ser160 for Cdk5; Ser18, Ser160 for ERK2, and Ser32 for PKA were identified by mass spectrometry. These sites were consistent with the bioinformatic predictions. The three N-terminal sites were also found to be phosphorylated in vivo in TPPP isolated from bovine brain. Affinity binding experiments provided evidence for the direct interaction between TPPP and ERK2. The phosphorylation of TPPP by ERK2 or Cdk5, but not by PKA, perturbed the structural alterations induced by the interaction between TPPP and tubulin without affecting the binding affinity (Kd = 2.5-2.7 μm) or the stoichiometry (1 mol TPPP/mol tubulin) of the complex. The phosphorylation by ERK2 or Cdk5 resulted in the loss of microtubule-assembling activity of TPPP. The combination of our in vitro and in vivo data suggests that ERK2 can regulate TPPP activity via the phosphorylation of Thr14 and/or Ser18 in its unfolded N-terminal tail.

Dephosphorylation of distinct sites on microtubule-associated protein MAP1B by protein phosphatases 1, 2A and 2B

Rat brain microtubule‐associated protein MAP1 B has been tested as a substrate for Ser/Thr protein phosphatases (PP). The dephosphorylation reactions were followed by specific antibodies recognizing phosphorylated and phosphorylatable epitopes. One set of phosphorylation sites on MAPI B are referred to as mode I sites, and their phosphorylation is presumably catalyzed by proline‐directed protein kinases. These mode I sites are efficiently dephosphorylated by PP2B and 2A but not by PP1. Another set of phosphorylation sites on MAP1B are named mode II sites, and their phosphorylation is possibly due to casein kinase II. These mode II sites are dephosphorylated by PP2A and PP1, the PP2B being ineffective. The selectivity of phosphatases for different sites within the same protein indicates the complexity of the dephosphorylation reactions regulating the functionality of MAP1B in neurons.

Protein phosphatase 2A is involved in hyphal growth of Neurospora crassa

Abstract Cantharidin and calyculin A, natural toxins that are inhibitors of protein phosphatases 1 and 2A (PP1 and PP2A, respectively), inhibit Neurospora crassa hyphal growth. When N. cassa was grown in the presence of either drug, abnormalities were observed at hyphal tips. In addition, both drugs induced an increase in hyphal branching. Cantharidin inhibited N. crassa hyphal growth in a temperature-dependent manner, as the effect of the drug was more pronounced at 34° C than at 25° C. In addition to the drug-mediated inhibition of phosphatase activity, a genetic approach was used to determine the phenotypic consequences of reduced PP2A activity. Two strains with subnormal PP2A activity were constructed. The first, in which the original pph-1 gene (encoding the PP2A catalytic subunit) was replaced with an ectopically integrated copy of pph-1, exhibited lower levels of pph-1 transcript, lower PP2A activity and increased sensitivity to cantharidin. Similarly, in a second strain, in which the pph-1 gene was cloned in an antisense orientation downstream of the inducible isocitrate lyase promoter, lower levels of pph-1 transcript, as well as of PP2A activity, and a reduction in hyphal growth were observed. The results of this study indicate that PP2A, and probably other Ser/Thr phosphatases, are involved in the regulation of hyphal growth in N. crassa.

Drosophila protein phosphatase V functionally complements a SIT4 mutant in Saccharomyces cerevisiae and its amino-terminal region can confer this complementation to a heterologous phosphatase catalytic domain.

The sequence of a Drosophila melanogaster cDNA encoding a novel 35 kDa protein serine/threonine phosphatase, termed PPV, is presented. PPV is 40‐41% identical to Drosophila PP1, 53% identical to Drosophila PP2A and 63% identical to Saccharomyces cerevisiae SIT4. Complementation studies demonstrated that PPV can functionally rescue a temperature sensitive mutant of SIT4, a protein phosphatase required for the G1 to S transition of the cell cycle. When placed under the SIT4 promoter, PPV cDNA is able to replace the SIT4 gene in S. cerevisiae. The amino‐terminal domain of PPV fused to another phosphatase catalytic region (PP1) also rescues the temperature sensitive SIT4 mutant and the SIT4 deletion mutant, implicating this region in binding to regulatory subunits and/or altering specificity. In Drosophila, a substantial transient increase in both PPV mRNA and protein occurs in late syncytial and early cellular blastoderm embryos. At the latter stage PPV is localized to the cytoplasm of cells at the cortex. This increase in PPV correlates with introduction of the G2 phase of the cell cycle, elevated zygotic transcription and cellularization, indicating that PPV may play a role in one or more of these processes.

Functional analysis of the Neurospora crassa PZL-1 protein phosphatase by expression in budding and fission yeast

The gene pzl‐1 from the filamentous fungus Neurospora crassa encodes a putative Ser/Thr protein phosphatase that is reminiscent of the Ppz1/Ppz2 and Pzh1 phosphatases from Saccharomyces cerevisiae and Schizosaccharomyces pombe, respectively. The entire PZL‐1 protein, as well as its carboxyl‐terminal domain, have been expressed in Escherichia coli as active protein phosphatases. To characterize its cellular role, PZL‐1 was also expressed in Sz. pombe and in S. cerevisiae. Expression of PZL‐1 in S. cerevisiae from the PPZ1 promoter was able to rescue the altered sensitivity to caffeine and lithium ions of a ppz1 strain. Furthermore, high copy number expression of PZL‐1 alleviated the lytic phenotype of a S. cerevisiae slt2/mpk1 mitogen‐activated protein (MAP) kinase mutant, similarly to that described for PPZ1, and mimicked the effects of high levels of Ppz1 on cell growth. Expression of PZL‐1 in fission yeast from a weak version of the nmt1 promoter fully rescued the growth defect of a pzh1Δ strain in high potassium, but only partially complemented the sodium‐hypertolerant phenotype. Strong overexpression of the N. crassa phosphatase in Sz. pombe affected cell growth and morphology. Therefore, PZL‐1 appears to fulfil every known function carried out by its S. cerevisiae counterpart, despite the marked divergence in sequence within their NH2‐terminal moieties. Copyright

Ribonucleoprotein-masked nicks at 50-kbp intervals in the eukaryotic genomic DNA

By using a microscopic approach, field inversion single-cell gel electrophoresis, we show that preformed single-strand discontinuities are present in the chromatin of resting and proliferating mammalian and yeast cells. These single-strand breaks are primarily nicks positioned at ≈50-kbp intervals throughout the entire genome that could be efficiently labeled in situ by DNA polymerase I holoenzyme but not by Klenow fragment and terminal transferase unless after ribonucleolytic treatments. The RNA molecules involved appear to comprise R-loops, recognized by the S9.6 RNA/DNA hybrid-specific antibody. By using the breakpoint cluster region of the Mixed Lineage Leukemia (MLL) gene as a model, we have found that the number of manifest nicks detected by FISH performed after field inversion single-cell gel electrophoresis depends on epigenetic context, but the difference between germ-line and translocated MLL alleles is abolished by protease treatment. Our data imply that the double-stranded genomic DNA is composed of contiguous rather than continuous single strands and reveal an aspect of higher-order chromatin organization with ribonucleoprotein-associated persistent nicks defining ≈50-kbp domains.

Differences in protein phosphorylation in vivo and in vitro between wild type and dunce mutant strains of Drosophila melanogaster

The protein phosphorylation patterns of wild type and dunce mutant strains of Drosophila melanogaster, as detected by sodium dodecylsulfate-gel electrophoresis and autoradiography, have been compared. After labelling in vivo with 32Pi or in vitro in homogenates with [gamma-32P]ATP, radioactive bands at and above apparent polypeptide mol. wt approximately 110,000 were more pronounced in dunce fly heads than in wild type heads. When labelling in vitro, in dunceM11 there appeared a radioactive band at apparent mol. wt approximately equal to 53,000 that was faintly visible in the wild strain. The same band could be intensified in both strains by adding cyclic AMP to the homogenate or by performing homogenization in the presence of theophylline. The data suggest that the mol. wt approximately equal to 53,000 protein is a substrate for cyclic AMP-dependent protein kinase.

Time-lapse video microscopy and image analysis of adherence and growth patterns of Candida albicans strains.

Digital image analysis of high time resolution video microscopy was used to investigate hyphal growth dynamics in different Candida albicans strains. The effects of the quorum sensing molecules tyrosol and farnesol, the deletion of the fungus specific protein phosphatase Z1 CaPPZ1), and the hypha-specific cyclin (HGC1) genes were analyzed by this method. Our system monitored cell growth in a CO2 incubator under near-physiological conditions and measured three major parameters under the following stringent conditions: (a) the time of yeast cell adherence, (b) the time of hyphal outgrowth, and (c) the rate of hyphal growth. This method showed that hyphal extension of wild-type SC5314 cells was accelerated by tyrosol and inhibited by farnesol. Hyphal growth rate was moderately lower in cappz1 and strongly reduced in hgc1 mutants. In addition, tyrosol treatment caused a firm adherence, while farnesol treatment and hgc1 mutation prevented the adherence of yeast cells to the surface of the culture flask. Transition from yeast-to-hyphal state was faster after tyrosol treatment, while it was reduced in farnesol-treated cells as well as in the cappz1 and hgc1 mutants. Our data confirm the notion that the attachment of yeast cells, the yeast-to-hyphal transition, and hyphal growth rate are closely related processes. Time-lapse video microscopy combined with image analysis offers a convenient and reliable method of testing chemicals, including potential drug candidates, and genetic manipulations on the dynamic morphological changes in C. albicans strains.

Protein phosphatase CaPpz1 is involved in cation homeostasis, cell wall integrity and virulence of Candida albicans

The opportunistic pathogen Candida albicans has a single protein phosphatase Z (PPZ) candidate gene termed CaPPZ1, which shows significant allele variability. We demonstrate here that bacterially expressed CaPpz1 protein exhibits phosphatase activity which can be inhibited by recombinant Hal3, a known inhibitor of Saccharomyces cerevisiae Ppz1. Site-directed mutagenesis experiments based on natural polymorphisms allowed the identification of three amino acid residues that affect enzyme activity or stability. The expression of CaPPZ1 in ppz1 S. cerevisiae and pzh1 Schizosaccharomyces pombe cells partially rescued the salt and caffeine phenotypes of the deletion mutants. CaPpz1 also complemented the slt2 S. cerevisiae mutant, which is crippled in the mitogen-activated protein (MAP) kinase that mediates the cell wall integrity signalling pathway. Collectively, our results suggest that the orthologous PPZ enzymes have similar but not identical functions in different fungi. The deletion of the CaPPZ1 gene in C. albicans resulted in a mutant that was sensitive to salts such as LiCl and KCl, to caffeine, and to agents that affect cell wall biogenesis such as Calcofluor White and Congo red, but was tolerant to spermine and hygromycin B. Reintegration of the CaPPZ1 gene into the deletion mutant alleviated all of the mutant phenotypes tested. Thus CaPpz1 is involved in cation homeostasis, cell wall integrity and the regulation of the membrane potential of C. albicans. In addition, the germ tube growth rate, and virulence in the BALB/c mouse model, were reduced in the null mutant, suggesting a novel function for CaPpz1 in the yeast to hypha transition that may have medical relevance.

Regulating the regulators. Conference on protein phosphorylation and protein phosphatases

The EMBO Conference on ‘Protein Phosphorylation and Protein Phosphatases’ was held in Marburg, Germany, July 8–12, 2001. The organizers were Susanne Klumpp (University of Marburg), Joaquin Arino (University of Barcelona) and Mathieu Bollen (University of Leuven). Next year the meeting will go to the USA. It will be organized by Ben Neel (bneel@caregroup.harvard.edu) and Angus Nairn and will be held at Snowmass, CO, July 13–18, 2002. The 2003 conference will be held in Europe again, at Sitges, near Barcelona, Spain, under the management of Joaquin Arino (joaquin.arino@uab.es), Susanne Klumpp and Denis Alexander. ![][1] According to a modest estimation, one third of the known eukaryotic proteins can be phosphorylated. Protein kinases are members of a large superfamily and incorporate the phosphate moiety into the serine (Ser), threonine (Thr) or tyrosine (Tyr) residues of their target proteins. In comparison, protein phosphatases are less numerous but more diverse. They can be classified into four independent protein families according to their substrate specificities, catalytic mechanisms and amino acid sequences (Figure 1). The phosphatases specific for Ser and Thr side chains were first identified in classical biochemical studies, but their subsequent cloning revealed significant structural differences between the family members, which led to their division into the phosphoprotein phosphatase (PPP) and metal‐ion‐dependent protein phosphatase (PPM) subfamilies. Molecular cloning and genetic studies also led to the discovery of the so‐called novel PPP enzymes. Despite the fact that both PPP and PPM subfamily members utilize the same binuclear metal assisted catalytic mechanism, they appear to have emerged through convergent evolution. Tyr‐specific protein phosphatases (PTPs) have been identified since and are composed of transmembrane (receptor‐like) and cytosolic (non‐receptor) subfamilies. These phosphatases not only counterbalance the effects of receptor and cytosolic protein Tyr kinases but also transmit information through the plasma membrane. Thus, they are of paramount importance in cellular … [1]: /embed/graphic-1.gif