Tight binding between a pool of the heterodimeric α/β tubulin and a protein kinase CK2 in<i>Trypanosoma cruzi</i>epimastigotes

Lima, Ana Rita de; Medina, Rafael; Uzcanga, Graciela L.; Noris-Suárez, Karem; Contreras, Víctor; Navarro, Marian; Arteaga, Rosa; Bubis, José

doi:10.1017/s0031182005009352

Cited by 12 publications

(8 citation statements)

References 49 publications

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“…It should be noted that ser/thr kinase CK2 was described to be tightly associated with the parasite heterodimeric α/β tubulins and capable of phosphorylating α and β tubulins in vitro [44],[45]. Moreover, phosphorylation of serine was identified in T. brucei β-tubulin (GL S VPELTQQMFDAK) [6].…”

Section: Discussionmentioning

confidence: 99%

Adhesion of Trypanosoma cruzi Trypomastigotes to Fibronectin or Laminin Modifies Tubulin and Paraflagellar Rod Protein Phosphorylation

et al. 2012

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BackgroundThe unicellular parasite Trypanosoma cruzi is the causative agent of Chagaś disease in humans. Adherence of the infective stage to elements of the extracellular matrix (ECM), as laminin and fibronectin, is an essential step in host cell invasion. Although members of the gp85/TS, as Tc85, were identified as laminin and fibronectin ligands, the signaling events triggered on the parasite upon binding to these molecules are largely unexplored.Methodology/Principal FindingsViable infective parasites were incubated with laminin, fibronectin or bovine serum albumin for different periods of time and the proteins were separated by bidimensional gels. The phosphoproteins were envisaged by specific staining and the spots showing phosphorylation levels significantly different from the control were excised and identified by MS/MS. The results of interest were confirmed by immunoblotting or immunoprecipitation and the localization of proteins in the parasite was determined by immunofluorescence. Using a host cell-free system, our data indicate that the phosphorylation contents of T. cruzi proteins encompassing different cellular functions are modified upon incubation of the parasite with fibronectin or laminin.Conclusions/SignificanceHerein it is shown, for the first time, that paraflagellar rod proteins and α-tubulin, major structural elements of the parasite cytoskeleton, are predominantly dephosphorylated during the process, probably involving the ERK1/2 pathway. It is well established that T. cruzi binds to ECM elements during the cell infection process. The fact that laminin and fibronectin induce predominantly dephosphorylation of the main cytoskeletal proteins of the parasite suggests a possible correlation between cytoskeletal modifications and the ability of the parasite to internalize into host cells.

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Section: Discussionmentioning

confidence: 99%

Adhesion of Trypanosoma cruzi Trypomastigotes to Fibronectin or Laminin Modifies Tubulin and Paraflagellar Rod Protein Phosphorylation

et al. 2012

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“…MS analysis of eIF5A purified from T. cruzi revealed two phosphorylation sites (Ser 2 and Tyr 21 ). Accordingly, Trypanosoma CK2 has been show to affect cellular growth, being involved in nucleocytoplasmic traffic [41] and α-tubulin phosphorylation [42]. Moreover, Ser 2 phosphorylation is also found in Trypanosoma brucei eIF5A phosphoproteome analyses [39], and has been detected in several lower eukaryotes and plants, suggesting a conserved role for this modification.…”

Section: Discussionmentioning

confidence: 99%

Eukaryotic initiation factor 5A dephosphorylation is required for translational arrest in stationary phase cells

Chung

Rocha

Tonelli

et al. 2013

Biochemical Journal

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The protein known as eIF5A (eukaryotic initiation factor 5A) has an elusive role in translation. It has a unique and essential hypusine modification at a conserved lysine residue in most eukaryotes. In addition, this protein is modified by phosphorylation with unknown functions. In the present study we show that a phosphorylated state of eIF5A predominates in exponentially growing Trypanosoma cruzi cells, and extensive dephosphorylation occurs in cells in stationary phase. Phosphorylation occurs mainly at Ser(2), as shown in yeast eIF5A. In addition, a novel phosphorylation site was identified at Tyr(21). In exponential cells, T. cruzi eIF5A is partially associated with polysomes, compatible with a proposed function as an elongation factor, and becomes relatively enriched in polysomal fractions in stationary phase. Overexpression of the wild-type eIF5A, or eIF5A with Ser(2) replaced by an aspartate residue, but not by alanine, increases the rate of cell proliferation and protein synthesis. However, the presence of an aspartate residue instead of Ser(2) is toxic for cells reaching the stationary phase, which show a less-pronounced protein synthesis arrest and a decreased amount of eIF5A in dense fractions of sucrose gradients. We conclude that eIF5A phosphorylation and dephosphorylation cycles regulate translation according to the growth conditions.

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“…New kinases which align with the casein kinase (CK) family often are catalytically active, as mentioned above, with casein isoforms and dephosphorylated casein [36, 42, 102-112]. In addition CK-specific substrates have been reported.…”

Section: Homology and Known Functional Substrates Of Homologuesmentioning

confidence: 99%

“…DDDEESITRR and KRRRAL(pS)VASLPGL [110] have also been used as CK1-specific peptide substrates, and RRREEE TEEE [110], RRREDEESDDEE, eIF2β-derived peptide MSGDEMIFDPTMSKKKKKKKKP [114] and RRASADDSDDEDL [115] have also been used as a CK2-specific peptide substrates. Typical Pep1 concentrations for activity assays are 100 – 800 μM [104-108, 113], while Pep2 concentrations usually range 40 -200 μM [33, 104–109, 112, 113]. Recombinant CK1.1 from T. cruzi had apparent Km for β-casein of 5.7 mg/mL and for Pep1 of 128 μM [108].…”

Section: Homology and Known Functional Substrates Of Homologuesmentioning

confidence: 99%

Enzyme Activity Assays for Protein Kinases: Strategies to Identify Active Substrates

Haubrich

Swinney²

2016

CDDT

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Protein kinases are an important class of enzymes and drug targets. New opportunities to discover medicines for neglected diseases can be leveraged by the extensive kinase tools and knowledge created in targeting human kinases. A valuable tool for kinase drug discovery is an enzyme assay that measures catalytic function. The functional assay can be used to identify inhibitors, estimate affinity, characterize molecular mechanisms of action (MMOAs) and evaluate selectivity. However, establishing an enzyme assay for a new kinases requires identification of a suitable substrate. Identification of a new kinase’s endogenous physiologic substrate and function can be extremely costly and time consuming. Fortunately, most kinases are promiscuous and will catalyze the phosphotransfer from ATP to alternative substrates with differing degrees of catalytic efficiency. In this manuscript we review strategies and successes in the identification of alternative substrates for kinases from organisms responsible for many of the neglected tropical diseases (NTDs) towards the goal of informing strategies to identify substrates for new kinases. Approaches for establishing a functional kinase assay include measuring auto-activation and use of generic substrates and peptides. The most commonly used generic substrates are casein, myelin basic protein, and histone. Sequence homology modeling can provide insights into the potential substrates and the requirement for activation. Empirical approaches that can identify substrates include screening of lysates (which may also help identify native substrates) and use of peptide arrays. All of these approaches have been used with a varying degree of success to identify alternative substrates.

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Tight binding between a pool of the heterodimeric α/β tubulin and a protein kinase CK2 inTrypanosoma cruziepimastigotes

Cited by 12 publications

References 49 publications

Adhesion of Trypanosoma cruzi Trypomastigotes to Fibronectin or Laminin Modifies Tubulin and Paraflagellar Rod Protein Phosphorylation

Adhesion of Trypanosoma cruzi Trypomastigotes to Fibronectin or Laminin Modifies Tubulin and Paraflagellar Rod Protein Phosphorylation

Eukaryotic initiation factor 5A dephosphorylation is required for translational arrest in stationary phase cells

Enzyme Activity Assays for Protein Kinases: Strategies to Identify Active Substrates

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