Mammary epithelial-specific knockout of the ephrin-B2 gene leads to precocious epithelial cell death at lactation

The family of Eph receptor tyrosine kinases and their membrane bound ligands, the ephrins, are involved in a wide variety of morphogenic processes during embryonic development and adult tissue homeostasis. Receptor-ligand interaction requires direct cell-cell contact and results in forward and reverse signaling originating from the receptor and ligand, respectively. We have previously shown that EphB4 and ephrinB2 are differentially expressed during the development of the adult mammary parenchyma. Overexpression of EphB4 in the mammary epithelium of transgenic mice leads to perturbations in mammary epithelial morphology, motility and growth. To investigate the role of ephrinB2 signaling in mammary gland biology, we have established transgenic mice exhibiting conditional ephrinB2 knockout in the mammary epithelium. In homozygote double transgenic CreLox mice, specific knockout of ephrinB2 occurred in the mammary epithelium during the first pregnancy-lactating period. Abolishing ephrinB2 function led to severe interference with the architecture and functioning of the mammary gland at lactation. The morphology of the transgenic lactating glands resembled that of involuting controls, with decreased epithelial cell number and collapsed lobulo-alveolar structures. Accordingly, massive epithelial cell death and expression of involution-specific genes were observed. Interestingly, in parallel to cell death, significant cell proliferation was apparent, suggestive of tissue regeneration.

Infection of primary canine duodenal epithelial cell cultures with Neospora caninum

According to current knowledge, sexual development of the apicomplexan parasite Neospora caninum takes place in the canine intestine. However, to date there is no information on the interaction between the parasite and the canine intestinal epithelium, and, next to the clinical and in vivo research tools, an in vitro model comprised of canine intestinal cells infected with N. caninum would be very helpful for investigations at the cellular level. Following the isolation of cells of neonatal canine duodenum and growth of cell cultures to monolayers for 5-6 days, canine intestinal epithelial cells were exposed to cell culture-derived N. caninum tachyzoites and bradyzoites. The host cells remained viable during in vitro culture for an average of 2 wk. During this time span, N. caninum was found to readily adhere to any surface area of these cells, but infection took mostly place at sites where microvilli-like structures were missing, e.g., at the cell periphery, with tachyzoites exhibiting at least 3-4 times increased invasive capacities compared to bradyzoites. Once intracellular, parasites resided within a parasitophorous vacuole, moved toward the vicinity of the nucleus and the more distal portion of the epithelial cells, and proliferated to form vacuoles of not more than 2-4 parasites, which were surrounded by numerous mitochondria. Immunofluorescence staining and TEM of infected cells showed that the expression of cytokeratins and the structural integrity of desmosomes and tight junctions were not notably altered during infection. Furthermore, no changes could be detected in the alkaline phosphatase activities in cell culture supernatants of infected and noninfected cells. Canine duodenal epithelial cell cultures represent a useful tool for future studies on the characteristics of the intestinal phases of N. caninum infection.

Interaction between Meristem Tissue Layers Controls Phyllotaxis

Phyllotaxis and vein formation are among the most conspicuous patterning processes in plants. The expression and polarization of the auxin efflux carrier PIN1 is the earliest marker for both processes, with mathematical models indicating that PIN1 can respond to auxin gradients and/or auxin flux. Here, we use cell-layer-specific PIN1 knockouts and partial complementation of auxin transport mutants to examine the interaction between phyllotactic patterning, which occurs primarily in the L1 surface layer of the meristem, and midvein specification in the inner tissues. We show that PIN1 expression in the L1 is sufficient for correct organ positioning, as long as the L1-specific influx carriers are present. Thus, differentiation of inner tissues can proceed without PIN1 or any of the known polar transporters. On theoretical grounds, we suggest that canalization of auxin flux between an auxin source and an auxin sink may involve facilitated diffusion rather than polar transport.

Sulfamethoxazole induces a switch mechanism in T cell receptors containing TCRVβ20-1, altering pHLA recognition

T cell receptors (TCR) containing Vβ20-1 have been implicated in a wide range of T cell mediated disease and allergic reactions, making it a target for understanding these. Mechanics of T cell receptors are largely unexplained by static structures available from x-ray crystallographic studies. A small number of molecular dynamic simulations have been conducted on TCR, however are currently lacking either portions of the receptor or explanations for differences between binding and non-binding TCR recognition of respective peptide-HLA. We performed molecular dynamic simulations of a TCR containing variable domain Vβ20-1, sequenced from drug responsive T cells. These were initially from a patient showing maculopapular eruptions in response to the sulfanilamide-antibiotic sulfamethoxazole (SMX). The CDR2β domain of this TCR was found to dock SMX with high affinity. Using this compound as a perturbation, overall mechanisms involved in responses mediated by this receptor were explored, showing a chemical action on the TCR free from HLA or peptide interaction. Our simulations show two completely separate modes of binding cognate peptide-HLA complexes, with an increased affinity induced by SMX bound to the Vβ20-1. Overall binding of the TCR is mediated through a primary recognition by either the variable β or α domain, and a switch in recognition within these across TCR loops contacting the peptide and HLA occurs when SMX is present in the CDR2β loop. Large binding affinity differences are induced by summed small amino acid changes primarily by SMX modifying only three critical CDR2β loop amino acid positions. These residues, TYRβ57, ASPβ64, and LYSβ65 initially hold hydrogen bonds from the CDR2β to adjacent CDR loops. Effects from SMX binding are amplified and traverse longer distances through internal TCR hydrogen bonding networks, controlling the overall TCR conformation. Thus, the CDR2β of Vβ20-1 acts as a ligand controlled switch affecting overall TCR binding affinity.

RP1 is a phosphorylation target of CK2 and is involved in cell adhesion

RP1 (synonym: MAPRE2, EB2) is a member of the microtubule binding EB1 protein family, which interacts with APC, a key regulatory molecule in the Wnt signalling pathway. While the other EB1 proteins are well characterized the cellular function and regulation of RP1 remain speculative to date. However, recently RP1 has been implicated in pancreatic cancerogenesis. CK2 is a pleiotropic kinase involved in adhesion, proliferation and anti-apoptosis. Overexpression of protein kinase CK2 is a hallmark of many cancers and supports the malignant phenotype of tumor cells. In this study we investigate the interaction of protein kinase CK2 with RP1 and demonstrate that CK2 phosphorylates RP1 at Ser(236) in vitro. Stable RP1 expression in cell lines leads to a significant cleavage and down-regulation of N-cadherin and impaired adhesion. Cells expressing a Phospho-mimicking point mutant RP1-ASP(236) show a marked decrease of adhesion to endothelial cells under shear stress. Inversely, we found that the cells under shear stress downregulate endogenous RP1, most likely to improve cellular adhesion. Accordingly, when RP1 expression is suppressed by shRNA, cells lacking RP1 display significantly increased cell adherence to surfaces. In summary, RP1 phosphorylation at Ser(236) by CK2 seems to play a significant role in cell adhesion and might initiate new insights in the CK2 and EB1 family protein association.

Phosphorylation of serine 4642 in the C-terminus of plectin by MNK2 and PKA modulates its interaction with intermediate filaments

Plectin is a versatile cytolinker of the plakin family conferring cell resilience to mechanical stress in stratified epithelia and muscles. It acts as a critical organizer of the cytoskeletal system by tethering various intermediate filament (IF) networks through its C-terminal IF-binding domain (IFBD). Mutations affecting the IFBD cause devastating human diseases. Here, we show that serine 4642, which is located in the extreme C-terminus of plectin, is phosphorylated in different cell lines. Phosphorylation of S4642 decreased the ability of plectin IFBD to associate with various IFs, as assessed by immunofluorescence microscopy and cell fractionation studies, as well as in yeast two-hybrid assays. Plectin phosphorylated at S4642 was reduced at sites of IF network anchorage along cell-substrate contacts in both skin and cultured keratinocytes. Treatment of SK-MEL-2 and HeLa cells with okadaic acid increased plectin S4642 phosphorylation, suggesting that protein phosphatase 2A dephosphorylates this residue. Moreover, plectin S4642 phosphorylation was enhanced after cell treatment with EGF, phorbol ester, sorbitol and 8-bromo-cyclic AMP, as well as during wound healing and protease-mediated cell detachment. Using selective protein kinase inhibitors, we identified two different kinases that modulate the phosphorylation of plectin S4642 in HeLa cells: MNK2, which is downstream of the ERK1/2-dependent MAPK cascade, and PKA. Our study indicates that phosphorylation of S4642 has an important regulatory role in the interaction of plectin with IFs and identifies a novel link between MNK2 and the cytoskeleton.

Clinical impact of programmed cell death ligand 1 expression in colorectal cancer

BACKGROUND Programmed cell death 1 (PD-1) receptor triggering by PD ligand 1 (PD-L1) inhibits T cell activation. PD-L1 expression was detected in different malignancies and associated with poor prognosis. Therapeutic antibodies inhibiting PD-1/PD-L1 interaction have been developed. MATERIALS AND METHODS A tissue microarray (n=1491) including healthy colon mucosa and clinically annotated colorectal cancer (CRC) specimens was stained with two PD-L1 specific antibody preparations. Surgically excised CRC specimens were enzymatically digested and analysed for cluster of differentiation 8 (CD8) and PD-1 expression. RESULTS Strong PD-L1 expression was observed in 37% of mismatch repair (MMR)-proficient and in 29% of MMR-deficient CRC. In MMR-proficient CRC strong PD-L1 expression correlated with infiltration by CD8(+) lymphocytes (P=0.0001) which did not express PD-1. In univariate analysis, strong PD-L1 expression in MMR-proficient CRC was significantly associated with early T stage, absence of lymph node metastases, lower tumour grade, absence of vascular invasion and significantly improved survival in training (P=0.0001) and validation (P=0.03) sets. A similar trend (P=0.052) was also detectable in multivariate analysis including age, sex, T stage, N stage, tumour grade, vascular invasion, invasive margin and MMR status. Interestingly, programmed death receptor ligand 1 (PDL-1) and interferon (IFN)-γ gene expression, as detected by quantitative reverse transcriptase polymerase chain reaction (RT-PCR) in fresh frozen CRC specimens (n=42) were found to be significantly associated (r=0.33, P=0.03). CONCLUSION PD-L1 expression is paradoxically associated with improved survival in MMR-proficient CRC.

A BAYESIAN APPROACH TO DOSE-RESPONSE ASSESSMENT AND DRUG-DRUG INTERACTION ANALYSIS: APPLICATION TO IN VITRO STUDIES

The considerable search for synergistic agents in cancer research is motivated by the therapeutic benefits achieved by combining anti-cancer agents. Synergistic agents make it possible to reduce dosage while maintaining or enhancing a desired effect. Other favorable outcomes of synergistic agents include reduction in toxicity and minimizing or delaying drug resistance. Dose-response assessment and drug-drug interaction analysis play an important part in the drug discovery process, however analysis are often poorly done. This dissertation is an effort to notably improve dose-response assessment and drug-drug interaction analysis. The most commonly used method in published analysis is the Median-Effect Principle/Combination Index method (Chou and Talalay, 1984). The Median-Effect Principle/Combination Index method leads to inefficiency by ignoring important sources of variation inherent in dose-response data and discarding data points that do not fit the Median-Effect Principle. Previous work has shown that the conventional method yields a high rate of false positives (Boik, Boik, Newman, 2008; Hennessey, Rosner, Bast, Chen, 2010) and, in some cases, low power to detect synergy. There is a great need for improving the current methodology. We developed a Bayesian framework for dose-response modeling and drug-drug interaction analysis. First, we developed a hierarchical meta-regression dose-response model that accounts for various sources of variation and uncertainty and allows one to incorporate knowledge from prior studies into the current analysis, thus offering a more efficient and reliable inference. Second, in the case that parametric dose-response models do not fit the data, we developed a practical and flexible nonparametric regression method for meta-analysis of independently repeated dose-response experiments. Third, and lastly, we developed a method, based on Loewe additivity that allows one to quantitatively assess interaction between two agents combined at a fixed dose ratio. The proposed method makes a comprehensive and honest account of uncertainty within drug interaction assessment. Extensive simulation studies show that the novel methodology improves the screening process of effective/synergistic agents and reduces the incidence of type I error. We consider an ovarian cancer cell line study that investigates the combined effect of DNA methylation inhibitors and histone deacetylation inhibitors in human ovarian cancer cell lines. The hypothesis is that the combination of DNA methylation inhibitors and histone deacetylation inhibitors will enhance antiproliferative activity in human ovarian cancer cell lines compared to treatment with each inhibitor alone. By applying the proposed Bayesian methodology, in vitro synergy was declared for DNA methylation inhibitor, 5-AZA-2'-deoxycytidine combined with one histone deacetylation inhibitor, suberoylanilide hydroxamic acid or trichostatin A in the cell lines HEY and SKOV3. This suggests potential new epigenetic therapies in cell growth inhibition of ovarian cancer cells.

Phosphorylation of the proline-rich domain of Xp95 modulates Xp95 interaction with partner proteins.

The mammalian adaptor protein Alix [ALG-2 (apoptosis-linked-gene-2 product)-interacting protein X] belongs to a conserved family of proteins that have in common an N-terminal Bro1 domain and a C-terminal PRD (proline-rich domain), both of which mediate partner protein interactions. Following our previous finding that Xp95, the Xenopus orthologue of Alix, undergoes a phosphorylation-dependent gel mobility shift during progesteroneinduced oocyte meiotic maturation, we explored potential regulation of Xp95/Alix by protein phosphorylation in hormone-induced cell cycle re-entry or M-phase induction. By MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS analyses and gel mobility-shift assays, Xp95 is phosphorylated at multiple sites within the N-terminal half of the PRD during Xenopus oocyte maturation, and a similar region in Alix is phosphorylated in mitotically arrested but not serum-stimulated mammalian cells. By tandem MS, Thr745 within this region, which localizes in a conserved binding site to the adaptor protein SETA [SH3 (Src homology 3) domain-containing, expressed in tumorigenic astrocytes] CIN85 (a-cyano-4-hydroxycinnamate)/SH3KBP1 (SH3-domain kinase-binding protein 1), is one of the phosphorylation sites in Xp95. Results from GST (glutathione S-transferase)-pull down and peptide binding/competition assays further demonstrate that the Thr745 phosphorylation inhibits Xp95 interaction with the second SH3 domain of SETA. However, immunoprecipitates of Xp95 from extracts of M-phase-arrested mature oocytes contained additional partner proteins as compared with immunoprecipitates from extracts of G2-arrested immature oocytes. The deubiquitinase AMSH (associated molecule with the SH3 domain of signal transducing adaptor molecule) specifically interacts with phosphorylated Xp95 in M-phase cell lysates. These findings establish that Xp95/Alix is phosphorylated within the PRD during M-phase induction, and indicate that the phosphorylation may both positively and negatively modulate their interaction with partner proteins.

ARTEMIS INTERACTS WITH THE CUL4A UBIQUITIN E3 LIGASE COMPLEX AND REGULATES THE CELL CYCLE PROGRESSION

Artemis, a member of the SNM1 gene family, is one of the six known components of the non-homologous end joining pathway. It is a multifunctional phospho-protein that has been shown to be modified by the phosphatidylinositol 3-kinases (PIKs) DNA-PKcs, ATM and ATR in response to a variety of cellular stresses. Artemis has important roles in V(D)J recombination, DNA double strand breaks repair and damage-induced cell-cycle checkpoint regulation. The detailed mechanism by which Artemis mediates its functions in these cellular pathways needs to be further elucidated. My work presented here demonstrates a new function for Artemis in cell cycle regulation as a component of Cullin-based E3 ligase complex. I show that Artemis interacts with Cul4A-DDB1 ligase complex via a direct interaction with the substrate-specific receptor DDB2, and deletion mapping analysis shows that part of the Snm1 domain of Artemis is responsible for this interaction. Additionally, Artemis also interacts with p27, a substrate of Cul4A-DDB1 complex, and both DDB2 and Artemis are required for the degradation of p27 mediated by this complex. Furthermore, I show that the regulation of p27 by Artemis and DDB2 is critical for cell cycle progression in normally proliferating cells and in response to serum withdrawal. Finally, I provide evidence showing that Artemis may be also a part of other Cullin-based E3 ligase complexes, and it has a role in controlling p27 levels in response to different cellular stress, such as UV irradiation. These findings suggest a novel pathway to regulate p27 protein level and define a new function for Artemis as an effector of Cullin-based E3-ligase mediated ubiquitylation, and thus, a cell cycle regulator in proliferating cells.

Inhibition of dendritic Ca2+ spikes by GABAB receptors in cortical pyramidal neurons is mediated by a direct Gi/o- -subunit interaction with Cav1 channels

Voltage-dependent calcium channels (VDCCs) serve a wide range of physiological functions and their activity is modulated by different neurotransmitter systems. GABAergic inhibition of VDCCs in neurons has an important impact in controlling transmitter release, neuronal plasticity, gene expression and neuronal excitability. We investigated the molecular signalling mechanisms by which GABAB receptors inhibit calcium-mediated electrogenesis (Ca2+ spikes) in the distal apical dendrite of cortical layer 5 pyramidal neurons. Ca2+ spikes are the basis of coincidence detection and signal amplification of distal tuft synaptic inputs characteristic for the computational function of cortical pyramidal neurons. By combining dendritic whole-cell recordings with two-photon fluorescence Ca2+ imaging we found that all subtypes of VDCCs were present in the Ca2+ spike initiation zone, but that they contribute differently to the initiation and sustaining of dendritic Ca2+ spikes. Particularly, Cav1 VDCCs are the most abundant VDCC present in this dendritic compartment and they generated the sustained plateau potential characteristic for the Ca2+ spike. Activation of GABAB receptors specifically inhibited Cav1 channels. This inhibition of L-type Ca2+ currents was transiently relieved by strong depolarization but did not depend on protein kinase activity. Therefore, our findings suggest a novel membrane-delimited interaction of the Gi/o-βγ-subunit with Cav1 channels identifying this mechanism as the general pathway of GABAB receptor-mediated inhibition of VDCCs. Furthermore, the characterization of the contribution of the different VDCCs to the generation of the Ca2+ spike provides new insights into the molecular mechanism of dendritic computation.

NETWORK TOPOLOGY IN HUMAN PROTEIN INTERACTION DATA PREDICTS FUNCTIONAL ASSOCIATION

High-throughput assays, such as yeast two-hybrid system, have generated a huge amount of protein-protein interaction (PPI) data in the past decade. This tremendously increases the need for developing reliable methods to systematically and automatically suggest protein functions and relationships between them. With the available PPI data, it is now possible to study the functions and relationships in the context of a large-scale network. To data, several network-based schemes have been provided to effectively annotate protein functions on a large scale. However, due to those inherent noises in high-throughput data generation, new methods and algorithms should be developed to increase the reliability of functional annotations. Previous work in a yeast PPI network (Samanta and Liang, 2003) has shown that the local connection topology, particularly for two proteins sharing an unusually large number of neighbors, can predict functional associations between proteins, and hence suggest their functions. One advantage of the work is that their algorithm is not sensitive to noises (false positives) in high-throughput PPI data. In this study, we improved their prediction scheme by developing a new algorithm and new methods which we applied on a human PPI network to make a genome-wide functional inference. We used the new algorithm to measure and reduce the influence of hub proteins on detecting functionally associated proteins. We used the annotations of the Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) as independent and unbiased benchmarks to evaluate our algorithms and methods within the human PPI network. We showed that, compared with the previous work from Samanta and Liang, our algorithm and methods developed in this study improved the overall quality of functional inferences for human proteins. By applying the algorithms to the human PPI network, we obtained 4,233 significant functional associations among 1,754 proteins. Further comparisons of their KEGG and GO annotations allowed us to assign 466 KEGG pathway annotations to 274 proteins and 123 GO annotations to 114 proteins with estimated false discovery rates of <21% for KEGG and <30% for GO. We clustered 1,729 proteins by their functional associations and made pathway analysis to identify several subclusters that are highly enriched in certain signaling pathways. Particularly, we performed a detailed analysis on a subcluster enriched in the transforming growth factor β signaling pathway (P<10-50) which is important in cell proliferation and tumorigenesis. Analysis of another four subclusters also suggested potential new players in six signaling pathways worthy of further experimental investigations. Our study gives clear insight into the common neighbor-based prediction scheme and provides a reliable method for large-scale functional annotations in this post-genomic era.

Physical and functional interaction between the dopamine transporter and the synaptic vesicle protein synaptogyrin-3.

Uptake through the dopamine transporter (DAT) represents the primary mechanism used to terminate dopaminergic transmission in brain. Although it is well known that dopamine (DA) taken up by the transporter is used to replenish synaptic vesicle stores for subsequent release, the molecular details of this mechanism are not completely understood. Here, we identified the synaptic vesicle protein synaptogyrin-3 as a DAT interacting protein using the split ubiquitin system. This interaction was confirmed through coimmunoprecipitation experiments using heterologous cell lines and mouse brain. DAT and synaptogyrin-3 colocalized at presynaptic terminals from mouse striatum. Using fluorescence resonance energy transfer microscopy, we show that both proteins interact in live neurons. Pull-down assays with GST (glutathione S-transferase) proteins revealed that the cytoplasmic N termini of both DAT and synaptogyrin-3 are sufficient for this interaction. Furthermore, the N terminus of DAT is capable of binding purified synaptic vesicles from brain tissue. Functional assays revealed that synaptogyrin-3 expression correlated with DAT activity in PC12 and MN9D cells, but not in the non-neuronal HEK-293 cells. These changes were not attributed to changes in transporter cell surface levels or to direct effect of the protein-protein interaction. Instead, the synaptogyrin-3 effect on DAT activity was abolished in the presence of the vesicular monoamine transporter-2 (VMAT2) inhibitor reserpine, suggesting a dependence on the vesicular DA storage system. Finally, we provide evidence for a biochemical complex involving DAT, synaptogyrin-3, and VMAT2. Collectively, our data identify a novel interaction between DAT and synaptogyrin-3 and suggest a physical and functional link between DAT and the vesicular DA system.

Endothelial cell-specific lymphotoxin-β receptor signaling is critical for lymph node and high endothelial venule formation.

The development of lymph nodes (LNs) and formation of LN stromal cell microenvironments is dependent on lymphotoxin-β receptor (LTβR) signaling. In particular, the LTβR-dependent crosstalk between mesenchymal lymphoid tissue organizer and hematopoietic lymphoid tissue inducer cells has been regarded as critical for these processes. Here, we assessed whether endothelial cell (EC)-restricted LTβR signaling impacts on LN development and the vascular LN microenvironment. Using EC-specific ablation of LTβR in mice, we found that conditionally LTβR-deficient animals failed to develop a significant proportion of their peripheral LNs. However, remnant LNs showed impaired formation of high endothelial venules (HEVs). Venules had lost their cuboidal shape, showed reduced segment length and branching points, and reduced adhesion molecule and constitutive chemokine expression. Due to the altered EC-lymphocyte interaction, homing of lymphocytes to peripheral LNs was significantly impaired. Thus, this study identifies ECs as an important LTβR-dependent lymphoid tissue organizer cell population and indicates that continuous triggering of the LTβR on LN ECs is critical for lymphocyte homeostasis.

Phosphorylation of the proline-rich domain of Xp95 modulates Xp95 interaction with partner proteins.

The mammalian adaptor protein Alix [ALG-2 (apoptosis-linked-gene-2 product)-interacting protein X] belongs to a conserved family of proteins that have in common an N-terminal Bro1 domain and a C-terminal PRD (proline-rich domain), both of which mediate partner protein interactions. Following our previous finding that Xp95, the Xenopus orthologue of Alix, undergoes a phosphorylation-dependent gel mobility shift during progesteroneinduced oocyte meiotic maturation, we explored potential regulation of Xp95/Alix by protein phosphorylation in hormone-induced cell cycle re-entry or M-phase induction. By MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS analyses and gel mobility-shift assays, Xp95 is phosphorylated at multiple sites within the N-terminal half of the PRD during Xenopus oocyte maturation, and a similar region in Alix is phosphorylated in mitotically arrested but not serum-stimulated mammalian cells. By tandem MS, Thr745 within this region, which localizes in a conserved binding site to the adaptor protein SETA [SH3 (Src homology 3) domain-containing, expressed in tumorigenic astrocytes] CIN85 (a-cyano-4-hydroxycinnamate)/SH3KBP1 (SH3-domain kinase-binding protein 1), is one of the phosphorylation sites in Xp95. Results from GST (glutathione S-transferase)-pull down and peptide binding/competition assays further demonstrate that the Thr745 phosphorylation inhibits Xp95 interaction with the second SH3 domain of SETA. However, immunoprecipitates of Xp95 from extracts of M-phase-arrested mature oocytes contained additional partner proteins as compared with immunoprecipitates from extracts of G2-arrested immature oocytes. The deubiquitinase AMSH (associated molecule with the SH3 domain of signal transducing adaptor molecule) specifically interacts with phosphorylated Xp95 in M-phase cell lysates. These findings establish that Xp95/Alix is phosphorylated within the PRD during M-phase induction, and indicate that the phosphorylation may both positively and negatively modulate their interaction with partner proteins.