A cell based screening approach for identifying protein degradation regulators.

Cellular transitions are achieved by the concerted actions of regulated degradation pathways. In the case of the cell cycle, ubiquitin mediated degradation ensures unidirectional transition from one phase to another. For instance, turnover of the cell cycle regulator cyclin B1 occurs after metaphase to induce mitotic exit. To better understand pathways controlling cyclin B1 turnover, the N-terminal domain of cyclin B1 was fused to luciferase to generate an N-cyclin B1-luciferase protein that can be used as a reporter for protein turnover. Prior studies demonstrated that cell-based screens using this reporter identified small molecules inhibiting the ubiquitin ligase controlling cyclin B1-turnover. Our group adapted this approach for the G2-M regulator Wee1 where a Wee1-luciferase construct was used to identify selective small molecules inhibiting an upstream kinase that controls Wee1 turnover. In the present study we present a screening approach where cell cycle regulators are fused to luciferase and overexpressed with cDNAs to identify specific regulators of protein turnover. We overexpressed approximately 14,000 cDNAs with the N-cyclin B1-luciferase fusion protein and determined its steady-state level relative to other luciferase fusion proteins. We identified the known APC/C regulator Cdh1 and the F-box protein Fbxl15 as specific modulators of N-cyclin B1-luciferase steady-state levels and turnover. Collectively, our studies suggest that analyzing the steady-state levels of luciferase fusion proteins in parallel facilitates identification of specific regulators of protein turnover.

Cell death induced by the Jak2 inhibitor, G6, correlates with cleavage of vimentin filaments.

Hyperkinetic Jak2 tyrosine kinase signaling has been implicated in several human diseases including leukemia, lymphoma, myeloma, and the myeloproliferative neoplasms. Using structure-based virtual screening, we previously identified a novel Jak2 inhibitor named G6. We showed that G6 specifically inhibits Jak2 kinase activity and suppresses Jak2-mediated cellular proliferation. To elucidate the molecular and biochemical mechanisms by which G6 inhibits Jak2-mediated cellular proliferation, we treated Jak2-V617F expressing human erythroleukemia (HEL) cells for 12 h with either vehicle control or 25 µM of the drug and compared protein expression profiles using two-dimensional gel electrophoresis. One differentially expressed protein identified by electrospray mass spectroscopy was the intermediate filament protein, vimentin. It was present in DMSO treated cells but absent in G6 treated cells. HEL cells treated with G6 showed both time- and dose-dependent cleavage of vimentin as well as a marked reorganization of vimentin intermediate filaments within intact cells. In a mouse model of Jak2-V617F mediated human erythroleukemia, G6 also decreased the levels of vimentin protein, in vivo. The G6-induced cleavage of vimentin was found to be Jak2-dependent and calpain-mediated. Furthermore, we found that intracellular calcium mobilization is essential and sufficient for the cleavage of vimentin. Finally, we show that the cleavage of vimentin intermediate filaments, per se, is sufficient to reduce HEL cell viability. Collectively, these results suggest that G6-induced inhibition of Jak2-mediated pathogenic cell growth is concomitant with the disruption of intracellular vimentin filaments. As such, this work describes a novel pathway for the targeting of Jak2-mediated pathological cell growth.

Phosphorylation of Y372 is critical for Jak2 tyrosine kinase activation.

Jak2 tyrosine kinase plays an important role in cytokine mediated signal transduction. There are 49 tyrosine residues in Jak2 and phosphorylation of some of these are known to play important roles in the regulation of Jak2 kinase activity. Here, using mass spectrometry, we identified tyrosine residues Y372 and Y373 as novel sites of Jak2 phosphorylation. Mutation of Y372 to F (Y372F) significantly inhibited Jak2 phosphorylation, including that of Y1007, whereas the Jak2-Y373F mutant displayed only modest reduction in phosphorylation. Relative to Jak2-WT, the ability of Jak2-Y372F to bind to and phosphorylate STAT1 was decreased, resulting in reduced Jak2-mediated downstream gene transcription. While the Y372F mutation had no effect on receptor-independent, hydrogen peroxide-mediated Jak2 activation, it impaired interferon-gamma (IFNγ) and epidermal growth factor (EGF)-dependent Jak2 activation. Interestingly however, the Y372F mutant exhibited normal receptor binding properties. Finally, co-expression of SH2-Bß only partially restored the activation of the Jak2-Y372F mutant suggesting that the mechanism whereby phosphorylation of Y372 is important for Jak2 activation is via dimerization. As such, our results indicate that Y372 plays a critical yet differential role in Jak2 activation and function via a mechanism involving Jak2 dimerization and stabilization of the active conformation.

Activation domain-dependent degradation of somatic Wee1 kinase.

Cell cycle progression is dependent upon coordinate regulation of kinase and proteolytic pathways. Inhibitors of cell cycle transitions are degraded to allow progression into the subsequent cell cycle phase. For example, the tyrosine kinase and Cdk1 inhibitor Wee1 is degraded during G(2) and mitosis to allow mitotic progression. Previous studies suggested that the N terminus of Wee1 directs Wee1 destruction. Using a chemical mutagenesis strategy, we report that multiple regions of Wee1 control its destruction. Most notably, we find that the activation domain of the Wee1 kinase is also required for its degradation. Mutations in this domain inhibit Wee1 degradation in somatic cell extracts and in cells without affecting the overall Wee1 structure or kinase activity. More broadly, these findings suggest that kinase activation domains may be previously unappreciated sites of recognition by the ubiquitin proteasome pathway.

Mapping the phosphorylation sites of Ulk1.

Ulk1 is a serine/threonine kinase that controls macroautophagy, an essential homeostatic recycling pathway that degrades bulk cytoplasmic material and directs the turnover of organelles such as peroxisomes and mitochondria. Further, macroautophagy is potently induced by signals that trigger metabolic stress, such as hypoxia and amino acid starvation, where its recycling functions provide macromolecules necessary to maintain catabolic metabolism and cell survival. Substrates for Ulk1 have not been identified, and little is known regarding post-translational control of Ulk1 kinase activity and function. To gain insights into the regulatory mechanisms of Ulk1, we developed a robust purification protocol for Ulk1 and demonstrated that Ulk1 is highly phosphorylated and requires autophosphorylation for stability. Importantly, high-resolution, tandem mass spectrometry identified multiple sites of phosphorylation on Ulk1, including several within domains known to regulate macroautophagy. Differential phosphorylation analyses also identified sites of phosphorylation in the C-terminal domain that depend upon or require Ulk1 autophosphorylation.

Proteomics of the nucleus ovoidalis and field L brain regions of zebra finch.

The purpose of present study is to analyze the brain proteome of the nucleus ovoidalis (OV) and Field L regions of the zebra finch (Taeniopygia guttata). The OV and Field L are important brain nuclei in song learning in zebra finches; their analyses identified a total of 79 proteins. The zebra finch brain proteome analyses are poised to provide clues about cell and circuit layout as well as possible circuit function.

Strategies to recover proteins from ocular tissues for proteomics.

We present here the results of protein extraction from different ocular regions using different detergents. Extraction strategies used to determine optimal protein extraction included: pressure cycling and aqueous-organic phase extraction in combination with electrophoretic fractionation for anterior, posterior, and peripapillary sclera. Detergent extraction of proteins from freshly enucleated porcine eyes (n = 8) showed significant differences for different eye regions. Protein yield ranged from 2.3 to 50.7 mug protein/mg for different ocular tissues, with the lens yielding the most protein. ASB-14 and Triton X-100 provided the best protein yields (n = 10) for anterior and posterior sclera. The spectrophotometric measurements for ASB-14 were not consistent with SDS-PAGE densitometry. A combination of 0.5% Triton X-100, 0.5% Tween-20, and 0.1% Genapol C-100 was found optimal for extraction from sclera. Proteins from different regions of the eye are best extracted with different detergents. The pressure cycling technology provided superior extraction compared to the other methods. Additional aqueous-organic phase partitioning enables superior fractionation when compared to SDS-PAGE alone. Organic phase fractionation is compatible with MS and allowed identification of 34, 71, and 77 proteins respectively from anterior, posterior, and peripapillary sclera. The extraction strategy may affect the final outcome in protein profiling by MS or by other methods.

The N-terminal SH2 domain of the tyrosine phosphatase, SHP-2, is essential for Jak2-dependent signaling via the angiotensin II type AT1 receptor.

Previous work has suggested that the protein tyrosine phosphatase, SHP-2, may act to facilitate angiotensin II (Ang II)-mediated, Jak2-dependent signaling. However, the mechanisms by which this occurs are not known. Here, Ang II-mediated, Jak2-dependent signaling was analyzed in a fibroblast cell line lacking the N-terminal, SH2 domain of SHP-2 (SHP-2(Delta46-110)). While the SHP-2(Delta46-110) cells were capable of activating Jak2 tyrosine kinase, they were unable to facilitate AT1 receptor/Jak2 co-association, STAT activation and subsequent Ang II-mediated gene transcription when compared to wild type control cells. These data therefore suggested that the N-terminal SH2 domain of SHP-2 was acting to recruit Jak2 to the AT1 receptor signaling complex. We found that the N-terminal SH2 domain of SHP-2 binds Jak2 predominantly, but not exclusively at tyrosine 201. Mass spectrometry analysis confirmed that this tyrosine residue is in fact phosphorylated. When this tyrosine was converted to phenylalanine, the ability of Jak2 to activate subsequent downstream signaling events was reduced. In summary, we have identified a novel site of Jak2 tyrosine autophosphorylation; namely, tyrosine 201. Our data suggest that the N-terminal SH2 domain of SHP-2 binds this amino acid residue. The functional consequence of this interaction is to recruit Jak2 to the AT1 receptor signaling complex and in turn promote downstream Jak2-dependent signaling.