Discovery of Potent Antimalarial Type II Kinase Inhibitors with Selectivity over Human Kinases.

While progress has been made in the effort to eradicate malaria, the disease remains a significant threat to global health. Acquired resistance to frontline treatments is emerging in Africa, urging a need for the development of novel antimalarial agents. Repurposing human kinase inhibitors provides a potential expedited route given the availability of a diverse array of kinase-targeting drugs that are approved or in clinical trials. Phenotypic screening of a library of type II human kinase inhibitors identified compound 1 as a lead antimalarial, which was initially developed to target human ephrin type A receptor 2 (EphA2). Here, we report a structure-activity relationship study and lead optimization of compound 1, which led to compound 33, with improved antimalarial activity and selectivity.

Differential Expression of miRNAs Contributes to Tumor Aggressiveness and Racial Disparity in African American Men with Prostate Cancer.

Prostate cancer is the leading cancer in incidence and second leading cause of cancer mortality in US men. African American men have significantly higher incidence and mortality rates from prostate cancer than European American men. Previous studies reported that the disparity in prostate cancer survival or mortality can be explained by different biological backgrounds. microRNAs (miRNAs) regulate gene expression of their cognate mRNAs in many cancers. Therefore, miRNAs may be a potentially promising diagnostic tool. The role of miRNAs in prostate cancer aggressiveness and racial disparity has not been fully established. The goal of this study is to identify miRNAs associated with aggressiveness and racial disparity in prostate cancer. Here we report miRNAs that are associated with tumor status and aggressiveness in prostate cancer using a profiling approach. Further, downregulated miRNAs in African American tissues were confirmed by qRT-PCR. These miRNAs have also been shown to negatively regulate the expression of the androgen receptor in prostate cancer cells. This report provides a novel insight into understanding tumor aggressiveness and racial disparities of prostate cancer.

Human Polo-like Kinase Inhibitors as Antiplasmodials.

Protein kinases have proven to be a very productive class of therapeutic targets, and over 90 inhibitors are currently in clinical use primarily for the treatment of cancer. Repurposing these inhibitors as antimalarials could provide an accelerated path to drug development. In this study, we identified BI-2536, a known potent human polo-like kinase 1 inhibitor, with low nanomolar antiplasmodial activity. Screening of additional PLK1 inhibitors revealed further antiplasmodial candidates despite the lack of an obvious orthologue of PLKs in Plasmodium. A subset of these inhibitors was profiled for their in vitro killing profile, and commonalities between the killing rate and inhibition of nuclear replication were noted. A kinase panel screen identified PfNEK3 as a shared target of these PLK1 inhibitors; however, phosphoproteome analysis confirmed distinct signaling pathways were disrupted by two structurally distinct inhibitors, suggesting PfNEK3 may not be the sole target. Genomic analysis of BI-2536-resistant parasites revealed mutations in genes associated with the starvation-induced stress response, suggesting BI-2536 may also inhibit an aminoacyl-tRNA synthetase.

Anticancer function of microRNA-30e is mediated by negative regulation of HELLPAR, a noncoding macroRNA, and genes involved in ubiquitination and cell cycle progression in prostate cancer.

Prostate cancer (PCa) progression relies on androgen receptor (AR) function, making AR a top candidate for PCa therapy. However, development of drug resistance is common, which eventually leads to development of castration-resistant PCa. This warrants a better understanding of the pathophysiology of PCa that facilitates the aberrant activation of key signaling pathways including AR. MicroRNAs (miRNAs) function as regulators of cancer progression as they modulate various cellular processes. Here, we demonstrate a multidimensional function of miR-30e through the regulation of genes involved in various signaling pathways. We noted loss of miR-30e expression in prostate tumors, which, when restored, led to cell cycle arrest, induction of apoptosis, improved drug sensitivity of PCa cells and reduced tumor progression in xenograft models. We show that experimental upregulation of miR-30e reduces expression of mRNAs including AR, FBXO45, SRSF7 and MYBL2 and a novel long noncoding RNA (lncRNA) HELLPAR, which are involved in cell cycle, apoptosis and ubiquitination, and the effects could be rescued by inhibition of miR-30e expression. RNA immunoprecipitation analysis confirmed direct interactions between miR-30e and its RNA targets. We noted a newly identified reciprocal relationship between miR-30e and HELLPAR, as inhibition of HELLPAR improved stabilization of miR-30e. Transcriptome profiling and quantitative real-time PCR (qRT-PCR) validation of miR-30e-expressing PCa cells showed differential expression of genes involved in cell cycle progression, apoptosis and ubiquitination, which supports our in vitro study. This study demonstrates an integrated function of miR-30e on dysregulation of miRNA/lncRNA/mRNA axes that may have diagnostic and therapeutic significance in aggressive PCa.

LncRNA PAINT is associated with aggressive prostate cancer and dysregulation of cancer hallmark genes.

Long noncoding RNAs (lncRNAs) play regulatory role in cellular processes and their aberrant expression may drive cancer progression. Here we report the function of a lncRNA PAINT (prostate cancer associated intergenic noncoding transcript) in promoting prostate cancer (PCa) progression. Upregulation of PAINT was noted in advanced stage and metastatic PCa. Inhibition of PAINT decreased cell proliferation, S-phase progression, increased expression of apoptotic markers, and improved sensitivity to docetaxel and Aurora kinase inhibitor VX-680. Inhibition of PAINT decreased cell migration and reduced expression of Slug and Vimentin. Ectopic expression of PAINT suppressed E-cadherin, increased S-phase progression and cell migration. PAINT expression in PCa cells induced larger colony formation, increased tumor growth and higher expression of mesenchymal markers. Transcriptome analysis followed by qRT-PCR validation showed differentially expressed genes involved in epithelial mesenchymal transition (EMT), apoptosis and drug resistance in PAINT-expressing cells. Our study establishes an oncogenic function of PAINT in PCa.

Dysregulation of DNA Methylation and Epigenetic Clocks in Prostate Cancer among Puerto Rican Men.

In 2021, approximately 248,530 new prostate cancer (PCa) cases are estimated in the United States. Hispanic/Latinos (H/L) are the second largest racial/ethnic group in the US. The objective of this study was to assess DNA methylation patterns between aggressive and indolent PCa along with ancestry proportions in 49 H/L men from Puerto Rico (PR). Prostate tumors were classified as aggressive (n = 17) and indolent (n = 32) based on the Gleason score. Genomic DNA samples were extracted by macro-dissection. DNA methylation patterns were assessed using the Illumina EPIC DNA methylation platform. We used ADMIXTURE to estimate global ancestry proportions. We identified 892 differentially methylated genes in prostate tumor tissues as compared with normal tissues. Based on an epigenetic clock model, we observed that the total number of stem cell divisions (TNSC) and stem cell division rate (SCDR) were significantly higher in tumor than adjacent normal tissues. Regarding PCa aggressiveness, 141 differentially methylated genes were identified. Ancestry proportions of PR men were estimated as African, European, and Indigenous American; these were 24.1%, 64.2%, and 11.7%, respectively. The identification of DNA methylation profiles associated with risk and aggressiveness of PCa in PR H/L men will shed light on potential mechanisms contributing to PCa disparities in PR population.

Influence of gene expression on survival of clear cell renal cell carcinoma.

Approximately 10%-20% of patients with clinically localized clear cell renal cell carcinoma (ccRCC) at time of surgery will subsequently experience metastatic progression. Although considerable progression was seen in the systemic treatment of metastatic ccRCC in last 20 years, once ccRCC spreads beyond the confines of the kidney, 5-year survival is less than 10%. Therefore, significant clinical advances are urgently needed to improve overall survival and patient care to manage the growing number of patients with localized ccRCC. We comprehensively evaluated expression of 388 candidate genes related with survival of ccRCC by using TCGA RNAseq (n = 515), Total Cancer Care (TCC) expression array data (n = 298), and a well characterized Moffitt RCC cohort (n = 248). We initially evaluated all 388 genes for association with overall survival using TCGA and TCC data. Eighty-one genes were selected for further analysis and tested on Moffitt RCC cohort using NanoString expression analysis. Expression of nine genes (AURKA, AURKB, BIRC5, CCNE1, MK167, MMP9, PLOD2, SAA1, and TOP2A) was validated as being associated with poor survival. Survival prognostic models showed that expression of the nine genes and clinical factors predicted the survival in ccRCC patients with AUC value: 0.776, 0.821 and 0.873 for TCGA, TCC and Moffitt data set, respectively. Some of these genes have not been previously implicated in ccRCC survival and thus potentially offer insight into novel therapeutic targets. Future studies are warranted to validate these identified genes, determine their biological mechanisms and evaluate their therapeutic potential in preclinical studies.

MicroRNAs as Guardians of the Prostate: Those Who Stand before Cancer. What Do We Really Know about the Role of microRNAs in Prostate Biology?

Prostate cancer is the second leading cause of cancer-related deaths of men in the Western world. Despite recent advancement in genomics, transcriptomics and proteomics to understand prostate cancer biology and disease progression, castration resistant metastatic prostate cancer remains a major clinical challenge and often becomes incurable. MicroRNAs (miRNAs), about 22-nucleotide-long non-coding RNAs, are a group of regulatory molecules that mainly work through post-transcriptional gene silencing via translational repression. Expression analysis studies have revealed that miRNAs are aberrantly expressed in cancers and have been recognized as regulators of prostate cancer progression. In this critical review, we provide an analysis of reported miRNA functions and conflicting studies as they relate to expression levels of specific miRNAs and prostate cancer progression; oncogenic and/or tumor suppressor roles; androgen receptor signaling; epithelial plasticity; and the current status of diagnostic and therapeutic applications. This review focuses on select miRNAs, highly expressed in normal and cancer tissue, to emphasize the current obstacles faced in utilizing miRNA data for significant impacts on prostate cancer therapeutics.

3D porous chitosan-chondroitin sulfate scaffolds promote epithelial to mesenchymal transition in prostate cancer cells.

Prostate cancer (PCa) is a common cancer in men that is curable prior to metastasis, when its prognosis worsens. Chondroitin sulfate (CS) is found in the extracellular matrix of normal prostate tissue and PCa, with greater content in metastatic PCa. Biomaterial scaffolds containing CS have yet to be evaluated for tumor microenvironment applications. Three-dimensional porous chitosan-CS (C-CS) scaffolds were developed and evaluated for PCa culture. Three C-CS scaffold compositions were prepared with 4 w/v% chitosan and 0.1, 0.5, and 1.0 w/v% CS and named 4-0.1, 4-0.5, and 4-1, respectively. The C-CS scaffolds had 90-95% porosity, average pore sizes between 143 and 166 µm, and no significant difference in scaffold stiffness. PC-3 and 22Rv1 PCa cells were cultured on the C-CS scaffolds to study the effect of CS on PCa growth and epithelial to mesenchymal transition (EMT). All C-CS scaffold compositions supported PCa growth and the 4-1 scaffolds had the greatest cell numbers for both PC-3 and 22Rv1. The C-CS scaffolds promoted upregulated EMT marker expression compared to 2D cultures with the greatest EMT marker expression in 4-1 scaffolds. Increasing CS concentration promoted upregulated vimentin expression in PC-3 cultures and N-cadherin and MMP-2 expression in 22Rv1 cultures. C-CS scaffolds promoted docetaxel drug resistance in PC-3 and 22Rv1 cultures and the 4-1 scaffold cultures had the greatest drug resistance. These results indicate that C-CS scaffolds are a promising in vitro platform for PCa.

Multifaceted Function of MicroRNA-299-3p Fosters an Antitumor Environment Through Modulation of Androgen Receptor and VEGFA Signaling Pathways in Prostate Cancer.

Prostate cancer (PCa) is one of the most common cancers to affect men worldwide. Androgen receptor (AR) signaling is central to PCa and PCa therapy. MicroRNAs (miRNAs) play crucial roles in the regulation of prostate cancer through modulation of signaling pathways. In the present study, we illustrate the functional significance and therapeutic benefit of miR-299-3p, an AR targeting microRNA, in PCa progression. We noted loss of expression of miR-299-3p in prostate tumors compared to noncancerous prostate tissues. Replenishment of miR-299-3p in C4-2B, 22Rv-1 and PC-3 cells contributed to cell cycle arrest, reduced proliferation, migration and increased expression of apoptotic markers. Additionally, overexpression of miR-299-3p induced a reduction of AR, PSA and VEGFA expression. AGO-RNA pulldown experiment showed enrichment of AR, VEGFA and miR-299-3p in C4-2B cells overexpressing miR-299-3p. miR-299-3p overexpression also inhibited epithelial mesenchymal transition, expression of Slug, TGF-ß3, phospho-AKT and phospho-PRAS40, but increased expression of E-cadherin. Furthermore, miR-299 overexpression resulted in reduced tumor growth in xenograft models and increased drug sensitivity. Overall, this study has identified novel mechanisms of antitumor and antimigration function of miR-299-3p through modulation of AR and VEGFA signaling pathways which lead to improved drug sensitivity of PCa.

FGD4 (Frabin) Overexpression in Pancreatic Neuroendocrine Neoplasms.

OBJECTIVE: The pathogenesis of pancreatic neuroendocrine tumors (PNETs) is still unclear. We propose Frabin as a new molecular alteration in PNETs. Frabin is a guanine nucleotide exchange factor playing a role in mediating actin cytoskeleton changes during cell migration, morphogenesis, polarization, and division. METHODS: Patients with PNETs of different grades were assessed for Frabin expression using immunohistochemistry and tissue microarray. The tissue microarray included 12 grade 1 and 3 grade 2 PNETs and 14 grade 3 pancreatic neuroendocrine carcinomas (PECAs). Frabin immunostain was scored with Allred system. Statistical analysis used SAS and R software. Immunohistochemistry scores were correlated with tumor grade and stage. The Spearman correlation coefficient was calculated with P values. RESULTS: Pancreatic neuroendocrine tumors were graded according to the World Health Organization 2017 guidelines. Frabin was expressed by 24 (82.7%) of the PNET/PECA studied. Only 5 (17.2%) of the 29 PNETs/PECA evaluated were Frabin negative. Frabin expression was cytoplasmic in all cases. We found a significant positive correlation (ρ = 0.47) between Frabin immunohistochemistry score and tumor grade (P = 0.01). No correlation was found between Frabin expression and tumor stage (P = 0.91). CONCLUSIONS: We report Frabin overexpression as a novel molecular alteration occurring in PNETs/PECAs.

3D porous chitosan-alginate scaffold stiffness promotes differential responses in prostate cancer cell lines.

Prostate cancer (PCa) is a leading cause of death for men worldwide. Most PCa patients die from metastasis and bone is the most common metastatic site. Three dimensional (3D) porous chitosan-alginate (CA) scaffolds were developed for bone tissue engineering and demonstrated for culture of cancer cells and enrichment of cancer stem cells. However, only a single scaffold composition was studied. Three compositions of 3D porous CA scaffolds (2, 4, and 6 wt%) were used to investigate the effect of scaffold stiffness on PCa cell response with PC-3, C4-2B, and 22Rv1 cell lines. The PC-3 cells formed cell clusters while the C4-2B and 22Rv1 cells formed multicellular spheroids. The three cell lines demonstrated stiffness independent cell growth and expressed phenotypic PCa biomarkers. The osteoblastic PCa lines C4-2B and 22Rv1 mineralized in basal media, while the osteolytic PC-3 line did not, demonstrating that CA scaffold cultures revealed differences in PCa phenotypes. The CA scaffolds are a 3D culture platform that supports PCa growth and phenotypic expression with adjustable scaffold stiffness to mimic stages of metastatic progression. Further investigation of the scaffolds for co-culture of PCa cells with fibroblasts and primary PCa cell culture should be conducted to develop a platform for screening chemotherapies.

Expression of FGD4 positively correlates with the aggressive phenotype of prostate cancer.

BACKGROUND: FGD4 (Frabin) is an F-actin binding protein with GTP/GDP exchange activity specific for CDC42. It is involved in reorganization of the actin cytoskeleton, which requires both actin binding and CDC42 activating function of FGD4. Expression of FGD4 is altered in patients with heterogeneous hereditary motor and sensory neuropathies as a result of demyelination of peripheral nerves. METHODS: In this study, we examined the expression of FGD4 in prostate cancer specimens using immunohistochemistry and studied the function of FGD4 in maintaining cell phenotype, behavior and drug sensitivity using overexpression and siRNA-based silencing approaches. We used Mann-Whitney test for comparative analysis of FGD4 expression. RESULTS: Our results show that the expression of FGD4 is upregulated in cancerous prostates compared to the luminal cells in benign prostatic hyperplasia, although the basal cells showed high staining intensities. We noted a gradual increase in the staining intensity of FGD4 with increasing aggressiveness of the disease. Inhibition of expression of FGD4 using siRNAs showed reduced proliferation and cell cycle arrest in G2/M phase of androgen dependent LNCaP-104S and androgen refractory PC-3 cells. Inhibition of FGD4 also resulted in reduced cell migration and CDC42 activities in PC-3 cells whereas, ectopic expression of FGD4 induced cell migration, altered expression of mesenchymal and epithelial markers and activation of CDC42/PAK signaling pathway. Reduced expression of FGD4 improved sensitivity of LNCaP-104S cells to the anti-androgen drug Casodex and PC-3 cells to the microtubule stabilizing drug docetaxel. CONCLUSIONS: Our data demonstrate a tumor promoting and a cell migratory function of FGD4 in prostate cancer cells and that inhibition of FGD4 expression enhances the response for both androgen-dependent and independent prostate cancer cells towards currently used prostate cancer drugs.

Characterization of Plasmodium falciparum Atypical Kinase PfPK7- Dependent Phosphoproteome.

PfPK7 is an "orphan" kinase displaying regions of homology to multiple protein kinase families. PfPK7 functions in regulating parasite proliferation/development as evident from the phenotype analysis of knockout parasites. Despite this regulatory role, the functions of PfPK7 in signaling pathways are not known. To better understand PfPK7-regulated phosphorylation events, we performed isobaric tag-based quantitative comparative phosphoproteomics of the schizont and segmenter stages from wild-type and pfpk7 - parasite lines. This analysis identified 3,875 phosphorylation sites on 1,047 proteins. Among these phosphorylation events, 146 proteins with 239 phosphorylation sites displayed reduction in phosphorylation in the absence of PfPK7. Further analysis of the phosphopeptides revealed three motifs whose phosphorylation was down regulated in the pfpk7 - cell line in both schizonts and segmenters. Decreased phosphorylation following loss of PfPK7 indicates that these proteins may function as direct substrates of PfPK7. We demonstrated that PfPK7 is active toward three of these potential novel substrates; however, PfPK7 did not phosphorylate many of the other proteins, suggesting that decreased phosphorylation in these proteins is an indirect effect. Our phosphoproteomics analysis is the first study to identify direct substrates of PfPK7 and reveals potential downstream or compensatory signaling pathways.

The other face of miR-17-92a cluster, exhibiting tumor suppressor effects in prostate cancer.

miR-17-92a cluster miRNAs are transcribed from a polycistronic transcription unit C13orf25 that generates six mature miRNAs, miR-17, miR-18a, miR-19a, miR-19b, miR-20a and miR-92a that are overexpressed in lung and colon cancers. Here we show that the expression of miR-17-92a miRNAs are reduced in cancerous prostate tissues compared to uninvolved areas and also in aggressive prostate cancer cells. Restoration of expression of all members of miR-17-92a cluster showed, decreased expression of cell cycle regulatory proteins cyclin D1 and SSH1; and LIMK1 and FGD4 of RhoGTPase signaling pathway. Expression of miR-17-92a miRNAs caused decreased cell proliferation, reduced activation of AKT and MAP kinases, delayed tumorigenicity and reduced tumor growth in animals. Expression of miR-17-92a miRNAs inhibited EMT via reduced cell migration and expression of mesenchymal markers while elevating expression and surface localization of the epithelial marker E-Cadherin. Expression of miR-17-92a miRNAs improved sensitivity of androgen dependent LNCaP 104-S prostate cancer cells to anti-androgen drug Casodex, AKT inhibitor MK-2206 2HCl, and docetaxel. The androgen refractory PC-3 cells also showed increased sensitivity to docetaxel, MK-2206 2HCl and Aurora kinase inhibitor VX680 upon ectopic expression of miR-17-92a cluster miRNAs. Our data demonstrate a tumor suppressor effect of miR-17-92a cluster miRNAs in prostate cancer cells and restoration of expression of these miRNAs has a therapeutic benefit for both androgen-dependent and -independent prostate cancer cells.

Aurora A kinase modulates actin cytoskeleton through phosphorylation of Cofilin: Implication in the mitotic process.

Aurora A kinase regulates early mitotic events through phosphorylation and activation of a variety of proteins. Specifically, Aur-A is involved in centrosomal separation and formation of mitotic spindles in early prophase. The effect of Aur-A on mitotic spindles is mediated by the modulation of microtubule dynamics and association with microtubule binding proteins. In this study we show that Aur-A exerts its effects on spindle organization through the regulation of the actin cytoskeleton. Aurora A phosphorylates Cofilin at multiple sites including S(3) resulting in the inactivation of its actin depolymerizing function. Aur-A interacts with Cofilin in early mitotic phases and regulates its phosphorylation status. Cofilin phosphorylation follows a dynamic pattern during the progression of prophase to metaphase. Inhibition of Aur-A activity induced a delay in the progression of prophase to metaphase. Aur-A inhibitor also disturbed the pattern of Cofilin phosphorylation, which correlated with the mitotic delay. Our results establish a novel function of Aur-A in the regulation of actin cytoskeleton reorganization, through Cofilin phosphorylation during early mitotic stages.

MicroRNA expressions associated with progression of prostate cancer cells to antiandrogen therapy resistance.

BACKGROUND: Development of resistance to androgen deprivation therapy (ADT) is a major obstacle for the management of advanced prostate cancer. Therapies with androgen receptor (AR) antagonists and androgen withdrawal initially regress tumors but development of compensatory mechanisms including AR bypass signaling leads to re-growth of tumors. MicroRNAs (miRNAs) are small regulatory RNAs that are involved in maintenance of cell homeostasis but are often altered in tumor cells. RESULTS: In this study, we determined the association of genome wide miRNA expression (1113 unique miRNAs) with development of resistance to ADT. We used androgen sensitive prostate cancer cells that progressed to ADT and AR antagonist Casodex (CDX) resistance upon androgen withdrawal and treatment with CDX. Validation of expression of a subset of 100 miRNAs led to identification of 43 miRNAs that are significantly altered during progression of cells to treatment resistance. We also show a correlation of altered expression of 10 proteins targeted by some of these miRNAs in these cells. CONCLUSIONS: We conclude that dynamic alterations in miRNA expression occur early on during androgen deprivation therapy, and androgen receptor blockade. The cumulative effect of these altered miRNA expression profiles is the temporal modulation of multiple signaling pathways promoting survival and acquisition of resistance. These early events are driving the transition to castration resistance and cannot be studied in already developed CRPC cell lines or tissues. Furthermore our results can be used a prognostic marker of cancers with a potential to be resistant to ADT.

In-fiber production of polymeric particles for biosensing and encapsulation.

Polymeric micro- and nanoparticles are becoming a mainstay in biomedicine, medical diagnostics, and therapeutics, where they are used in implementing sensing mechanisms, as imaging contrast agents, and in drug delivery. Current approaches to the fabrication of such particles are typically finely tuned to specific monomer or polymer species, size ranges, and structures. We present a general scalable methodology for fabricating uniformly sized spherical polymeric particles from a wide range of polymers produced with complex internal architectures and continuously tunable diameters extending from the millimeter scale down to 50 nm. Controllable access to such a wide range of sizes enables broad applications in cancer treatment, immunology, and vaccines. Our approach harnesses thermally induced, predictable fluid instabilities in composite core/cladding polymer fibers drawn from a macroscopic scaled-up model called a "preform." Through a stack-and-draw process, we produce fibers containing a multiplicity of identical cylindrical cores made of the polymers of choice embedded in a polymer cladding. The instability leads to the breakup of the initially intact cores, independent of the polymer chemistry, into necklaces of spherical particles held in isolation within the cladding matrix along the entire fiber length. We demonstrate here surface functionalization of the extracted particles for biodetection through specific protein-protein interactions, volumetric encapsulation of a biomaterial in spherical polymeric shells, and the combination of both surface and volumetric functionalities in the same particle. These particles used in distinct modalities may be produced from the desired biocompatible polymer by changing only the geometry of the macroscopic preform from which the fiber is drawn.

Global analysis of protein expression and phosphorylation of three stages of Plasmodium falciparum intraerythrocytic development.

During asexual intraerythrocytic development, Plasmodium falciparum diverges from the paradigm of the eukaryotic cell cycles by undergoing multiple rounds of DNA replication and nuclear division without cytokinesis. A better understanding of the molecular switches that coordinate a myriad of events for the progression of the parasite through the intraerythrocytic developmental stages will be of fundamental importance for rational design of intervention strategies. To achieve this goal, we performed isobaric tag-based quantitative proteomics and phosphoproteomics analyses of three developmental stages in the Plasmodium asexual cycle and identified 2767 proteins, 1337 phosphoproteins, and 6293 phosphorylation sites. Approximately 34% of identified proteins and 75% of phosphorylation sites exhibit changes in abundance as the intraerythrocytic cycle progresses. Our study identified 43 distinct phosphorylation motifs and a range of potential MAPK/CDK substrates. Further analysis of phosphorylated kinases identified 30 protein kinases with 126 phosphorylation sites within the kinase domain or in N- or C-terminal tails. Many of these phosphorylations are likely CK2-mediated. We define the constitutive and regulated expression of the Plasmodium proteome during the intraerythrocytic developmental cycle, offering an insight into the dynamics of phosphorylation during asexual cycle progression. Our system-wide comprehensive analysis is a major step toward defining kinase-substrate pairs operative in various signaling networks in the parasite.

A functional cooperativity between Aurora A kinase and LIM kinase1: implication in the mitotic process.

Aurora kinase A (Aur-A), a mitotic kinase, regulates initiation of mitosis through centrosome separation and proper assembly of bipolar spindles. LIM kinase 1 (LIMK1), a modulator of actin and microtubule dynamics, is involved in the mitotic process through inactivating phosphorylation of cofilin. Phosphorylated LIMK1 is recruited to the centrosomes during early prophase, where it colocalizes with γ-tubulin. Here, we report a novel functional cooperativity between Aur-A and LIMK1 through mutual phosphorylation. LIMK1 is recruited to the centrosomes during early prophase and then to the spindle poles, where it colocalizes with Aur-A. Aur-A physically associates with LIMK1 and activates it through phosphorylation, which is important for its centrosomal and spindle pole localization. Aur-A also acts as a substrate of LIMK1, and the function of LIMK1 is important for its specific localization and regulation of spindle morphology. Taken together, the novel molecular interaction between these two kinases and their regulatory roles on one another's function may provide new insight on the role of Aur-A in manipulation of actin and microtubular structures during spindle formation.