Systematic and quantitative analysis of protein phosphorylation is revealing dynamic regulatory networks underlying cellular responses to environmental cues. However, matching these sites to the kinases that phosphorylate them and the phosphorylation-dependent binding domains that may subsequently bind to them remains a challenge. NetPhorest is an atlas of consensus sequence motifs that covers 179 kinases and 104 phosphorylation-dependent binding domains [Src homology 2 (SH2), phosphotyrosine binding (PTB), BRCA1 C-terminal (BRCT), WW, and 14–3–3]. The atlas reveals new aspects of signaling systems, including the observation that tyrosine kinases mutated in cancer have lower specificity than their non-oncogenic relatives. The resource is maintained by an automated pipe line, which uses phylogenetic trees to structure the currently available in vivo and in vitro data to derive probabilistic sequence models of linear motifs. The atlas is available as a community resource (http://netphorest.info).

Cell fate can be determined by asymmetric segregation of gene expression regulators. In the budding yeast Saccharomyces cerevisiae, the transcription factor Ace2 accumulates specifically in the daughter cell nucleus, where it drives transcription of genes that are not expressed in the mother cell. The NDR/LATS family protein kinase Cbk1 is required for Ace2 segregation and function. Using peptide scanning arrays, we determined Cbk1′s phosphorylation consensus motif, the first such unbiased approach for an enzyme of this family, showing that it is a basophilic kinase with an unusual preference for histidine −5 to the phosphorylation site. We found that Cbk1 phosphorylates such sites in Ace2, and that these modifications are critical for Ace2′s partitioning and function. Using proteins marked with GFP variants, we found that Ace2 moves from isotropic distribution to the daughter cell nuclear localization, well before cytokinesis, and that the nucleus must enter the daughter cell for Ace2 accumulation to occur. We found that Cbk1, unlike Ace2, is restricted to the daughter cell. Using both in vivo and in vitro assays, we found that two critical Cbk1 phosphorylations block Ace2′s interaction with nuclear export machinery, while a third distal modification most likely acts to increase the transcription factor's activity. Our findings show that Cbk1 directly controls Ace2, regulating the transcription factor's activity and interaction with nuclear export machinery through three phosphorylation sites. Furthermore, Cbk1 exhibits a novel specificity that is likely conserved among related kinases from yeast to metazoans. Cbk1 is functionally restricted to the daughter cell, and cannot diffuse from the daughter to the mother. In addition to providing a mechanism for Ace2 segregation, these findings show that an isotropically distributed cell fate determinant can be asymmetrically partitioned in cytoplasmically contiguous cells through spatial segregation of a regulating protein kinase.

Polo-like kinase-1 (Plk1) phosphorylates a number of mitotic substrates, but the diversity of Plk1-dependent processes suggests the existence of additional targets. Plk1 contains a specialized phosphoserine-threonine binding domain, the Polo-box domain (PBD), postulated to target the kinase to its substrates. Using the specialized PBD of Plk1 as an affinity capture agent, we performed a screen to define the mitotic Plk1-PBD interactome by mass spectrometry. We identified 622 proteins that showed phosphorylation-dependent mitosis-specific interactions, including proteins involved in well-established Plk1-regulated processes, and in processes not previously linked to Plk1 such as translational control, RNA processing, and vesicle transport. Many proteins identified in our screen play important roles in cytokinesis, where, in mammalian cells, the detailed mechanistic role of Plk1 remains poorly defined. We go on to characterize the mitosis-specific interaction of the Plk1-PBD with the cytokinesis effector kinase Rho-associated coiled-coil domain-containing protein kinase 2 (Rock2), demonstrate that Rock2 is a Plk1 substrate, and show that Rock2 colocalizes with Plk1 during cytokinesis. Finally, we show that Plk1 and RhoA function together to maximally enhance Rock2 kinase activity in vitro and within cells, and implicate Plk1 as a central regulator of multiple pathways that synergistically converge to regulate actomyosin ring contraction during cleavage furrow ingression.

The timing and localization of events during mitosis is controlled by the regulated phosphorylation of proteins by the mitotic kinases, which include Aurora A, Aurora B, Nek2, Plk1, and the cyclin-dependent kinase complex Cdk1/cyclin B. Although mitotic kinases can have overlapping subcellular localizations, each kinase appears to phosphorylate its substrates on distinct sites. To gain insight into the relative importance of local sequence context in kinase selectivity, identify previously unknown substrates of these five mitotic kinases, and explore potential mechanisms for substrate discrimination, we determined the optimal substrate motifs of these major mitotic kinases by Positional Scanning Oriented Peptide Library Screening (PS-OPLS). We verified individual motifs with in vitro peptide kinetic studies and used structural modeling to rationalize the kinase-specific selection of key motif-determining residues at the molecular level. Cross comparisons among the phosphorylation site selectivity motifs of these kinases revealed an evolutionarily conserved mutual exclusion mechanism in which the positively and negatively selected portions of the phosphorylation motifs of mitotic kinases, together with their subcellular localizations, result in proper substrate targeting in a coordinated manner during mitosis.

Orthotopic liver transplantation (OLT) is the only effective treatment for end-stage liver disease due to primary sclerosing cholangitis (PSC)., and steroid-resistant ACR (29% versus 0%; P ‫؍‬ 0.012). Despite the strong linkage disequilibrium between DRB1*08 and DQB1*04, DRB1*08-positive subjects with recurrence were negative for DQB1*04, whereas the single DRB1*08-positive subject without recurrent PSC was positive for DQB1*04. A history of ACR and presence of HLA-DRB1*08 are associated with increased risk of recurrent PSC, suggesting an immunologic mechanism for this syndrome. Further studies are required to confirm these observations and to understand the underlying mechanisms.

Invasive fungal infection is a serious complication of orthotopic liver transplantation, but its risk factors remain incompletely defined. Iron overload has already been associated with increased risk of fungal infections, but it has not yet been assessed as a risk factor in liver transplantation. We retrospectively studied a cohort of 153 consecutive patients who underwent their first liver transplantation at a single center and who survived at least 7 days after transplantation. The association between various pretransplant patient characteristics, including hepatic explant iron and risk of invasive fungal infections, was analyzed by univariate and multivariate models. Iron in the hepatic explant was assessed by Perl's Prussian blue stain by a pathologist blinded to clinical outcome. During the first year after transplantation, 28 of 153 patients developed a total of 31 invasive fungal infections, of which 21 (68%) were caused by Candida, 7 (23%) by Aspergillus, 2 (6%) by Cryptococcus, and 1 (3%) by Saccharomyces. Stainable iron in the hepatic explant was found in 48 patients (31%). Stainable iron in the hepatic explant was found to be strongly and independently associated with posttransplantation fungal infections in multivariate analysis (hazard ratio 3.09; 95% confidence interval 1.45-6.56; P ϭ 0. See Editorial on Page 1731Invasive fungal infection is a serious complication of liver transplantation, developing in 5-42% of liver transplant recipients, with a mortality rate of 10-75% for candidiasis and 80-100% for aspergillosis. 1 Several baseline and posttransplantation variables have been identified as risk factors of invasive fungal infections. These include retransplantation, abdominal surgery after transplantation, a lengthy transplant operation, class 2 human leukocyte antigen mismatch, fulminant hepatic failure, renal dysfunction, dialysis, hyperglycemia requiring insulin therapy, posttransplantation bacterial infection, symptomatic cytomegalovirus infection, prolonged stay in the intensive care unit, prolonged or multiple antibiotic therapy, multiple blood or plasma transfusions, and low pretransplantation serum albumin levels. 2-9 Although models have been developed to stratify transplant recipients according to risk of developing invasive fungal infections, 1,3 there remains considerable scope for defining other potential risk factors for invasive fungal infections in liver transplant recipients. Identification of additional risk factors might allow for more rational targeted antifungal prophylaxis and/or closer monitoring for invasive fungal infections.There are several lines of evidence suggesting that iron overload might predispose to invasive fungal infections. First, iron is known to be an essential growth factor for many pathogenic fungi, and limiting iron availability to medically important fungi can render an Abbreviation: MELD, Model for End-Stage Liver Disease.

Taken together, these findings suggest a strong association between fatty liver disease and HCC in non-cirrhotic livers.

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