An analysis of phosphorylation sites in protein kinases from Leishmania

Protein kinases are promising drug targets for Leishmaniasis. We have evaluated the phosphorylation potential of protein kinases in different species and strains of Leishmania. Phosphorylation potential of serine, threonine and tyrosine residues of kinases in Leishmania parasite were studied. The results indicate that some species specific residues of serine, threonine and tyrosine have a phosphorylation potential of 1 suggesting that these residues are important target sites in protein kinases based anti-leishmanial therapies.

Protein kinases act on serine, threonine, tyrosine, or histidine residues of proteins leading to phosphorylation. Phosphorylation can modify the function of a protein in a variety of ways. It can alter activity of a protein, stabilize it or cause degradation, localize it within a particular compartment of a cell, can initiate or disrupt its interaction with other proteins. The protein kinases make up the majority of all kinases and are widely studied. These kinases along with phosphatases, play a major role in protein and enzyme regulation along with signaling in the cell [3].
Adaptation to changes in the environment is usually initiated by extracellular signals that regulate the transcription of specific genes [4]. The transduction of signals to the nucleus of eukaryotic cells is mainly mediated by phosphorylation cascades through the activity of serine/threonine and tyrosine kinases. The central role of protein phosphorylation in signal transduction, cellular transformation and differentiation in eukaryotic systems has become well established. Complex networks of protein phosphorylation/dephosphorylation represent important mechanisms in cellular regulation and transformation. Protein phosphorylation at tyrosine appears to be common signaling mechanism in eukaryotes [4]. As in higher eukaryotes, it is likely that protozoans control a variety of intracellular events through the reversible phosphorylation of proteins. Understanding of phosphorylation and dephosphorylation events are of considerable interest, but are relatively unexplored in Leishmania donovani. Lopes and colleagues have provided evidence of an infection-enhancing role of PKR in L. amazonensis infections. L. amazonensis infection of cultured human or mouse macrophage cell lines induced the activation of PKR as monitored by the time-dependent increase in the phosphorylated form of PKR (pPKR) [5]. In cells infected by pathogens, there is an increasing appreciation that dysregulated expression of protein kinases promotes the success of intracellular infections. In the current study we have deduced the phosphorylation potential of serine, threonine and tyrosine residues of protein kinases of different species and strains of Leishmania.

Methodology: Dataset creation
In this study, we aligned the protein sequences of Protein kinase of Leishmania spp associated with Leishmaniasis under similar prediction conditions and compared the results obtained. The protein sequences were procured from the National Center for Biotechnology Information (NCBI) through their Entrez search. gi_731707594 Leishmania panamensis 24 10 6 10 gi_7658170 Leishmania major 4 0 0  Table 1 for further details). Dendrogram A dendrogram (from Greek dendron "tree", -gramma"drawing") is a tree diagram used to illustrate the arrangement of the clusters produced by a clustering algorithm.

Phylogeny
A phylogenetic tree or evolutionary tree shows the evolutionary relationships among various biological species or other entities that are believed to have a common ancestor. In a phylogenetic tree, each node with descendants represented the most recent common ancestor of the descendants, and the edge lengths in some trees correspond to time estimates. We referred each node as a taxonomic unit. Internal nodes are generally called hypothetical taxonomic units (HTUs) as they cannot be directly observed.

Phosphorylation potential
A number of in silico tools have been developed in order to predict plausible phosphorylation sites in a given protein, lipid, carbohydrate, DNA or RNA. An attempt in this regard was made by [6] whereby they conducted a benchmark study including the leading protein phosphorylation prediction software, in an effort to determine which performs best. NetPhos 2.0 predicted all phosphorylation sites with fidelity of 69%. So we performed prediction of phosphorylation sites using Net Phos 2.0.
Protein sequences of various species and strains expressing protein kinases having different accession nos were obtained from NCBI and further subjected to analysis of phosphorylation sites using Net Phos 2.0 input sequences must be in the one-letter code. Peptides shorter than 9 residues can produce non-reliable results. So it's always advisable to include 4 residues on both sides of the Tyr/Ser/Thr to be evaluated. The optimal is to use the complete "native" protein sequence. By default the server uses protein kinase specificity prediction without any filtering.
NetPhos is a neural network-based method for predicting potential phosphorylation sites at serine, threonine or tyrosine residues in protein sequences. The first version of NetPhos (v.1.0) was developed in 1997-1998 and was only available for in-house use and research collaborations. Version 2.0 was trained on a larger data set of known phosphorylation sites and has been made publicly available using the WWW or via e-mail. Protein phosphorylation at serine, threonine or tyrosine residues affect a multitude of cellular signaling processes. Here we study specificity in substrate recognition and phosphorylation by protein kinases. Net Phos is an artificial neural network method that predicts phosphorylation sites in independent sequences with sensitivity in the range from 69% to 96%.

Discussion:
Leishmania activated Kinases (LACK) play an important role in parasite thermo tolerance and virulence [7]. Stage specific protein phosphorylation patterns [8] and an extensive examination of phosphorylation changes during promastigote-to-amastigote differentiation [9] have been recently described. Phylogeny analysis (Figure 1) shows evolutionary relationship of different species strains of protein kinase of Leishmania associated with Leishmaniasis. gi |731707594| and gi|146085061| are closely related. gi| 389603714|, gi|685203703| and gi|731695473| are closely related. Similarly gi|322498592|, gi|134069253| are closely related as compared to gi|401421203|. gi|68126239| is the most distant of all followed by gi | 7658170|. Protein kinases gi_14608506 and gi_73170759 show higher number of serine and threonine residues while no of tyrosine residues are higher in the former strain. While the other strains like gi_13406925, gi_32249859, gi_38960371, gi_40142120, gi_68126239, gi_68520370, gi_73169547 have reasonable higher no of serine, threonine and tyrosines. gi_7658170 has only 4 residues of serine without any threonine and tyrosines which indicates lower potential of phosphorylation. In gi_14608506 (Figure 2A)

Conclusion:
The study reveals that serine, threonine and tyrosine residues of protein kinases having phosphorylation potential close to 1 could be an important target for anti-leishmanial therapies. Hence, different species strains of Leishmania provide important insights into development of anti-leishmanial drug regimes using kinase inhibitors.