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Delineation of the complement of proteins comprising the zygote and ookinete, the early developmental stages of Plasmodium within the mosquito midgut, is fundamental to understand initial molecular parasite-vector interactions. The published proteome of Plasmodium falciparum does not include analysis of the zygote/ookinete stages, nor does that of P. berghei include the zygote stage or secreted proteins. P. gallinaceum zygote, ookinete, and ookinete-secreted/released protein samples were prepared and subjected to Multidimensional protein identification technology (MudPIT). Peptides of P. gallinaceum zygote, ookinete, and ookinete-secreted proteins were identified by MS/MS, mapped to ORFs (>50 amino acids) in the extent P. gallinaceum whole genome sequence, and then matched to homologous ORFs in P. falciparum. A total of 966 P. falciparum ORFs encoding orthologous proteins were identified; just over 40% of these predicted proteins were found to be hypothetical. A majority of putative proteins with predicted secretory signal peptides or transmembrane domains were hypothetical proteins. This analysis provides a more comprehensive view of the hitherto unknown proteome of the early mosquito midgut stages of P. falciparum. The results underpin more robust study of Plasmodium-mosquito midgut interactions, fundamental to the development of novel strategies of blocking malaria transmission.

Regulation of the actin-myosin cytoskeleton plays a central role in cell migration and cancer progression. Here, we report the discovery of a cytoskeleton-associated kinase, pseudopodium-enriched atypical kinase 1 (PEAK1). PEAK1 is a 190-kDa nonreceptor tyrosine kinase that localizes to actin filaments and focal adhesions. PEAK1 undergoes Src-induced tyrosine phosphorylation, regulates the p130Cas-Crk-paxillin and Erk signaling pathways, and operates downstream of integrin and epidermal growth factor receptors (EGFR) to control cell spreading, migration, and proliferation. Perturbation of PEAK1 levels in cancer cells alters anchorage-independent growth and tumor progression in mice. Notably, primary and metastatic samples from colon cancer patients display amplified PEAK1 levels in 81% of the cases. Our findings indicate that PEAK1 is an important cytoskeletal regulatory kinase and possible target for anticancer therapy.

Because the mammalian target of rapamycin (mTOR) pathway is commonly deregulated in human cancer, mTOR inhibitors, rapamycin and its derivatives, are being actively tested in cancer clinical trials. Clinical updates indicate that the anticancer effect of these drugs is limited, perhaps due to rapamycin-dependent induction of oncogenic cascades by an as yet unclear mechanism. As such, we investigated rapamycin-dependent phosphoproteomics and discovered that 250 phosphosites in 161 cellular proteins were sensitive to rapamycin. Among these, rapamycin regulated four kinases and four phosphatases. A siRNA-dependent screen of these proteins showed that AKT induction by rapamycin was attenuated by depleting cellular CDC25B phosphatase. Rapamycin induces the phosphorylation of CDC25B at Serine375, and mutating this site to Alanine substantially reduced CDC25B phosphatase activity. Additionally, expression of CDC25B (S375A) inhibited the AKT activation by rapamycin, indicating that phosphorylation of CDC25B is critical for CDC25B activity and its ability to transduce rapamycin-induced oncogenic AKT activity. Importantly, we also found that CDC25B depletion in various cancer cell lines enhanced the anticancer effect of rapamycin. Together, using rapamycin phosphoproteomics, we not only advance the global mechanistic understanding of the action of rapamycin but also show that CDC25B may serve as a drug target for improving mTOR-targeted cancer therapies. [Cancer Res 2009;69(6):OF2663–8]

Immobilized metal affinity chromatography (IMAC) is a common strategy used for the enrichment of phosphopeptides from digested protein mixtures. However, this strategy by itself is inefficient when analyzing complex protein mixtures. Here, we assess the effectiveness of using protein-based IMAC as a pre-enrichment step prior to peptide-based IMAC. Ultimately, we couple the two IMAC-based enrichments and MudPIT in a quantitative phosphoproteomic analysis of the epidermal growth factor pathway in mammalian cells identifying 4470 unique phosphopeptides containing 4729 phosphorylation sites.

Deregulated Her2/ErbB2 receptor tyrosine kinase drives tumorigenesis and tumor progression in a variety of human tissues. Her2 transmits oncogenic signals through phosphorylation of its cytosolic domain. To study innate cellular mechanisms for containing Her2 oncogenic phosphorylation, a siRNA phosphatase library was screened for cellular phosphatase(s) that enhance phosphorylation in the signaling motif of Her2 after knockdown. We found that silencing protein tyrosine phosphatase PTPN13 significantly augmented growth factor-induced phosphorylation of the Her2 signaling domain and promoted the invasiveness of Her2-deregulated cancer cells. In addition, we discovered that growth factor-induced phosphorylation of PTPN13 was essential for the dephosphorylation of Her2 suggesting a negative feedback mechanism induced by growth factor to inhibit cellular Her2 activity through PTPN13. Importantly, we showed that PTPN13 mutations previously reported in human tumors significantly reduced the phosphatase activity of PTPN13, and consequently elevated the oncogenic potential of Her2 and the invasiveness of Her2-overexpressing human cancer cells. Taken together, these results suggest that cellular PTPN13 inhibits Her2 activity by dephosphorylating the signal domain of Her2 and plays a role in attenuating invasiveness and metastasis of Her2 overactive tumors.

Sur2 is a metazoan Mediator subunit that interacts with the adenovirus E1A protein and functions in a mitogen-activated protein kinase pathway required for vulva development in Caenorhabditis elegans . We generated sur2 −/− embryonic stem cells to analyze its function as a mammalian Mediator component. Our results show that Sur2 forms a subcomplex of the Mediator with two other subunits, TRAP/Med100 and 95. Knock-out of Sur2 prevents activation by E1A-CR3 and the mitogen-activated protein kinase–regulated ETS transcription factor Elk-1, but not by multiple other transcription factors. These results imply that specific activation domains stimulate transcription by binding to distinct Mediator subunits. Activation by E1A and Elk-1 requires recruitment of Mediator to a promoter by binding to its Sur2 subunit.

The Saccharomyces cerevisiae Mediator complex was originally identified because of its ability to stimulate activated transcription in vitro. Many of the subunits of the purified Mediator complex (24) are encoded by SRB genes, first characterized as suppressors of a deletion in the C-terminal heptapeptide repeat (CTD) of the large subunit of RNA polymerase II (Pol II) (23). Additional Mediator subunits are encoded by genes initially identified in other genetic screens for mutations affecting gene control and are named accordingly (e.g., RGR-1). Mediator subunits not characterized previously were called Med1, Med2, etc. (24). Mediator subunits in yeast have also been purified as part of a still larger holoenzyme complex including Pol II and several general transcription factors (GTFs) (23). The Pol II holoenzyme analyzed by Young and colleagues (see reference 23 for a review) includes Srb8, Srb9, Srb10, and Srb11, whereas the Mediator complex studied by Myers et al. (24) lacks these subunits. The Srb8 to Srb11 subunits form a functional subcomplex of the holoenzyme required for repression by several yeast repressors (3, 11, 23). These subunits are regulated differently from other yeast Mediator and holoenzyme subunits. The intracellular Srb10 concentration falls dramatically as yeast cells deplete nutrients from their media, whereas the concentrations of other Mediator subunits do not (11). Recently, Liu et al. analyzed yeast Mediator complexes in a nuclear extract, avoiding ion-exchange chromatography and high salt concentration to avoid dissociation of subunits (18). Under these conditions they found that the majority of each Mediator subunit, including Srb8 to Srb11, was associated with Pol II in a complex of ∼1.9 MDa that lacks GTFs. A less abundant complex of ∼0.55 MDa included a subset of Mediator subunits. Several mammalian multiprotein complexes have been identified that have several subunits homologous to components of the yeast Mediator and several subunits that are not clearly related to yeast proteins (21). Broadly speaking, two size classes of complexes have been identified. Complexes of ∼2 MDa, such as the TRAP/SMCC (12), NAT (32), DRIP (30), ARC (25), and human Mediator (2) complexes, share an overlapping set of components. Smaller complexes (∼500 to 700 kDa) containing Srb/Med homologs have also been identified, including the murine Mediator (13), CRSP (31), and PC2 (20) complexes. These mammalian Mediator-like complexes were identified and purified by different biochemical procedures. TRAP (7), DRIP (30), ARC (25), and human Mediator (2) were purified on the basis of their ability to bind to activation domains during affinity chromatography. SMCC (8) and NAT (32) were purified based on their content of CDK8, which is a homolog of yeast Srb10. Functions of these Mediator complexes were assayed in different in vitro transcription systems that varied in the purity of the GTFs and the use of naked DNA versus chromatin templates. Most of these complexes, including ARC, DRIP, PC2, and CRSP, greatly stimulated activated transcription (21). NAT and SMCC repressed activated transcription in assays with highly purified factors (8, 32), but SMCC activated transcription when TFIIH was omitted (8). This repression has been attributed to the phosphorylation of the cyclin H subunit of TFIIH by the CDK8 kinase within the Mediator complexes (1). The human Mediator complex inhibited activated transcription in a highly purified system but stimulated high levels of activated transcription in reactions with partially purified GTFs (2). While the different mammalian Mediator-like complexes so far described share many subunits, they also differ with regard to their reported subunit composition (21). This raises the question whether there are multiple distinct Mediator-like complexes in mammalian cells that may differ in their functional properties or whether there is in fact one or a small number of mammalian Mediator complexes. In the latter case, the apparent differences in subunit composition reported by different laboratories might result from relatively minor differences in the methods used to characterize the subunits or from different methods of purification that partially dissociate a single large complex. To estimate the number of different complexes containing Mediator subunits in HeLa cells, we subjected unfractionated HeLa cell nuclear extract to gel filtration chromatography at low salt concentration to avoid the dissociation of subunits. Protein complexes in eluted fractions were characterized by immunoblotting with several antibodies specific for Mediator subunits, including components of both size classes of Mediator complexes described. A single peak of ∼2 MDa containing each of the several Mediator subunits was observed. No significant peak was observed at ∼500 to 700 kDa, indicating that either this size class of Mediator complex is much less abundant than the ∼2-MDa size class or that the ∼500 to 700-kDa Mediator complexes described above were derived from the larger size class by dissociation during the multiple steps of column chromatography used in their purification. Mediator subunits CDK8, cyclin C, and hSur2 were also observed in lower-molecular-mass complexes, but only the ∼2-MDa size class significantly stimulated activated transcription. High-resolution gel filtration and immunoprecipitation analyses indicated that there are at least two subclasses of ∼2-MDa Mediator complexes, one containing CDK8 and cyclin C and one lacking these subunits. A total of ∼300,000 hSur2 subunits per cell were present in the ∼2-MDa Mediator complexes; this number is approximately equal to the number of Pol II molecules per HeLa cell (15).

We describe a genome-wide functional assay for rapid isolation of cell clones and genetic elements responsive to specific stimuli. A promoterless beta-lactamase reporter gene was transfected into a human T-cell line to generate a living library of reporter-tagged clones. When loaded with a cell-permeable fluorogenic substrate, the cell library simultaneously reports the expression of a large number of endogenous genes. Flow cytometry was used to recover individual clones whose reporter-tagged genes were either induced or repressed following T-cell activation. Responsive clones were expanded and analyzed pharmacologically to identify patterns of regulation associated with specific genes. Although demonstrated using T cells, the genomic assay could be applied to map downstream transcriptional consequences for any propagating cell line in response to any stimulus of interest.