Juan S. Martinez

Acm1 Is a Negative Regulator of the Cdh1-Dependent Anaphase-Promoting Complex/Cyclosome in Budding Yeast

ABSTRACT Cdh1 is a coactivator of the anaphase-promoting complex/cyclosome (APC/C) and contributes to mitotic exit and G1 maintenance by facilitating the polyubiquitination and subsequent proteolysis of specific substrates. Here, we report that budding yeast Cdh1 is a component of a cell cycle-regulated complex that includes the 14-3-3 homologs Bmh1 and Bmh2 and a previously uncharacterized protein, which we name Acm1 (APC/CCdh1modulator 1). Association of Cdh1 with Bmh1 and Bmh2 requires Acm1, and the Acm1 protein is cell cycle regulated, appearing late in G1 and disappearing in late M. In acm1Δ strains, Cdh1 localization to the bud neck and association with two substrates, Clb2 and Hsl1, were strongly enhanced. Several lines of evidence suggest that Acm1 can suppress APC/CCdh1-mediated proteolysis of mitotic cyclins. First, overexpression of Acm1 fully restored viability to cells expressing toxic levels of Cdh1 or a constitutively active Cdh1 mutant lacking inhibitory phosphorylation sites. Second, overexpression of Acm1 was toxic in sic1Δ cells. Third, ACM1 deletion exacerbated a low-penetrance elongated-bud phenotype caused by modest overexpression of Cdh1. This bud elongation was independent of the morphogenesis checkpoint, and the combination of acm1Δ and hsl1Δ resulted in a dramatic enhancement of bud elongation and G2/M delay. Effects on bud elongation were attenuated when Cdh1 was replaced with a mutant lacking the C-terminal IR dipeptide, suggesting that APC/C-dependent proteolysis is required for this phenotype. We propose that Acm1 and Bmh1/Bmh2 constitute a specialized inhibitor of APC/CCdh1.

Cdc14 phosphatases preferentially dephosphorylate a subset of cyclin-dependent kinase (Cdk) sites containing phosphoserine.

Background: Cdc14 phosphatases help control mitosis by dephosphorylating sites (Ser/Thr-Pro) targeted by cyclin-dependent kinases (Cdks). Results: Cdc14 family phosphatases strongly prefer phosphoserine over phosphothreonine at Cdk sites. Conclusion: By discriminating among Cdk sites, Cdc14 may participate in setting the order and timing of Cdk substrate dephosphorylation. Significance: Mechanisms governing the timing of Cdk site dephosphorylation are crucial for proper coordination of late mitotic events. Mitotic cell division is controlled by cyclin-dependent kinases (Cdks), which phosphorylate hundreds of protein substrates responsible for executing the division program. Cdk inactivation and reversal of Cdk-catalyzed phosphorylation are universal requirements for completing and exiting mitosis and resetting the cell cycle machinery. Mechanisms that define the timing and order of Cdk substrate dephosphorylation remain poorly understood. Cdc14 phosphatases have been implicated in Cdk inactivation and are thought to be generally specific for Cdk-type phosphorylation sites. We show that budding yeast Cdc14 possesses a strong and unusual preference for phosphoserine over phosphothreonine at Pro-directed sites in vitro. Using serine to threonine substitutions in the Cdk consensus sites of the Cdc14 substrate Acm1, we demonstrate that phosphoserine specificity exists in vivo. Furthermore, it appears to be a conserved property of all Cdc14 family phosphatases. An invariant active site residue was identified that sterically restricts phosphothreonine binding and is largely responsible for phosphoserine selectivity. Optimal Cdc14 substrates also possessed a basic residue at the +3 position relative to the phosphoserine, whereas substrates lacking this basic residue were not effectively hydrolyzed. The intrinsic selectivity of Cdc14 may help establish the order of Cdk substrate dephosphorylation during mitotic exit and contribute to roles in other cellular processes.

Phosphorylation Assay Based on Multifunctionalized Soluble Nanopolymer

Quantitative phosphorylation analysis is essential to understanding cellular signal transductions. Here we present a novel technology for the highly efficient assay of protein phosphorylation in high-throughput format without the use of phospho-specific antibodies. The technique is based on a water-soluble, nanosize polymer, termed pIMAGO, that is multifunctionalized with titanium(IV) ions for specific binding to phosphoproteins and with biotin groups that allow for enzyme-linked spectrometric detection. The sensitivity, specificity, and quantitative nature of pIMAGO for phosphorylation assays were examined with standard phosphoproteins and with purified phosphoproteins from whole cell extracts. As low as 100 pg of phosphoprotein can be measured quantitatively with the pIMAGO chemiluminescence assay. The pIMAGO assay was applied to an in vitro kinase assay, kinase inhibitor screening, and measurement of endogenous phosphorylation events. The technique provides a universal, quantitative method for global phosphorylation analysis with high sensitivity and specificity.

Drosophila activated Cdc42 kinase has an anti-apoptotic function.

Activated Cdc42 kinases (Acks) are evolutionarily conserved non-receptor tyrosine kinases. Activating somatic mutations and increased ACK1 protein levels have been found in many types of human cancers and correlate with a poor prognosis. ACK1 is activated by epidermal growth factor (EGF) receptor signaling and functions to regulate EGF receptor turnover. ACK1 has additionally been found to propagate downstream signals through the phosphorylation of cancer relevant substrates. Using Drosophila as a model organism, we have determined that Drosophila Ack possesses potent anti-apoptotic activity that is dependent on Ack kinase activity and is further activated by EGF receptor/Ras signaling. Ack anti-apoptotic signaling does not function through enhancement of EGF stimulated MAP kinase signaling, suggesting that it must function through phosphorylation of some unknown effector. We isolated several putative Drosophila Ack interacting proteins, many being orthologs of previously identified human ACK1 interacting proteins. Two of these interacting proteins, Drk and yorkie, were found to influence Ack signaling. Drk is the Drosophila homolog of GRB2, which is required to couple ACK1 binding to receptor tyrosine kinases. Drk knockdown blocks Ack survival activity, suggesting that Ack localization is important for its pro-survival activity. Yorkie is a transcriptional co-activator that is downstream of the Salvador-Hippo-Warts pathway and promotes transcription of proliferative and anti-apoptotic genes. We find that yorkie and Ack synergistically interact to produce tissue overgrowth and that yorkie loss of function interferes with Ack anti-apoptotic signaling. Our results demonstrate how increased Ack signaling could contribute to cancer when coupled to proliferative signals.

Drosophila Vap-33 Is Required for Axonal Localization of Dscam Isoforms

Mutations in VAPB have been identified in a familial form of amyotrophic lateral sclerosis (ALS), and reduced VAPB levels have been found in patients with sporadic ALS. Vap protein family members from different species and cell types have been implicated in a number of cellular functions, but how Vap dysfunction in neurons and/or muscles contributes to motor neuron degeneration and death is poorly understood. Using Drosophila as a model organism, we show that Vap physically interacts with and affects the axonal functions of the Down syndrome cell adhesion molecule (Dscam). Dscam is a cell-surface receptor involved in axon and dendritic patterning and neuron self-recognition and avoidance. Alternative splicing of the Dscam transcript leads to the production of Dscam isoforms that contain one of two possible transmembrane (TM) domain and flanking sequences that either restrict the isoform to dendrites and cell bodies (TM1) or target the isoform to axon processes (TM2). We find that Vap specifically interacts with Dscam isoforms that contain the TM2 cytoplasmic juxtamembrane flanking sequences. Using loss-of-function genetics, we further show that Vap is required for localization of Dscam isoforms containing TM2 to axons and that Vap loss suppresses Dscam gain-of-function axon phenotypes. We propose that Vap function is required in neurons to selectively traffic proteins to axons, and disruption of this function may contribute to the pathology of ALS.

A general strategy for studying multisite protein phosphorylation using label-free selected reaction monitoring mass spectrometry

The majority of eukaryotic proteins are phosphorylated in vivo, and phosphorylation may be the most common regulatory posttranslational modification. Many proteins are phosphorylated at numerous sites, often by multiple kinases, which may have different functional consequences. Understanding biological functions of phosphorylation events requires methods to detect and quantify individual sites within a substrate. Here we outline a general strategy that addresses this need and relies on the high sensitivity and specificity of selected reaction monitoring (SRM) mass spectrometry, making it potentially useful for studying in vivo phosphorylation without the need to isolate target proteins. Our approach uses label-free quantification for simplicity and general applicability, although it is equally compatible with stable isotope quantification methods. We demonstrate that label-free SRM-based quantification is comparable to conventional assays for measuring the kinetics of phosphatase and kinase reactions in vitro. We also demonstrate the capability of this method to simultaneously measure relative rates of phosphorylation and dephosphorylation of substrate mixtures, including individual sites on intact protein substrates in the context of a whole cell extract. This strategy should be particularly useful for characterizing the physiological substrate specificity of kinases and phosphatases and can be applied to studies of other protein modifications as well.

Acm1 contributes to nuclear positioning by inhibiting Cdh1-substrate interactions

The anaphase-promoting complex (APC) is tightly regulated during cell division, often by pseudosubstrate binding to its coactivators Cdh1 and Cdc20. Budding yeast Acm1 is a Cdh1 pseudosubstrate inhibitor whose biological function is unknown. We show here that cells lacking Acm1 have defects in nuclear positioning and spindle morphology during mitosis. However, Cdh1 substrates are not destabilized in the absence of Acm1 and expression of inactive Cdh1 mutants that retain substrate binding is sufficient for the acm1 phenotype. We conclude that Acm1 is not required to inhibit APCCdh1 activity but rather prevents untimely Cdh1-substrate interactions. We further provide evidence suggesting that the substrate primarily responsible for the acm1 phenotype is the bud neck-localized kinase, Hsl1. Our results imply that at least some coactivator-substrate interactions require regulation. Several unrelated APC pseudosubstrates have been identified in diverse eukaryotes and their ability to simultaneously inhibit enzymatic activity and substrate binding may partly explain why this regulatory mechanism has been selected repeatedly during evolution.

Molding BRCA2 function through its interacting partners

The role of the tumor suppressor BRCA2 has been shaped over 2 decades thanks to the discovery of its protein and nucleic acid partners, biochemical and structural studies of the protein, and the functional evaluation of germline variants identified in breast cancer patients. Yet, the pathogenic and functional effect of many germline mutations in BRCA2 remains undetermined, and the heterogeneity of BRCA2-associated tumors challenges the identification of causative variants that drive tumorigenesis. In this review, we propose an overview of the established and emerging interacting partners and functional pathways attributed to BRCA2, and we speculate on how variants altering these functions may contribute to cancer susceptibility.

A Comparative Study of Phoneme- and Word-Based Learning of English Words Presented to the Skin

Past research has demonstrated that speech communication on the skin is entirely achievable. However, there is still no definitive conclusion on the best training method that minimizes the time it takes for users to reach a prescribed performance level with a speech communication device. The present study reports the design and testing of two learning approaches with a system that translates English phonemes to haptic stimulation patterns (haptic symbols). With the phoneme-based learning approach, users learned the haptic symbols associated with the phonemes before attempting to acquire words made up of the phonemes. With the word-based approach, users learned words on day one. Two experiments were conducted with the two learning approaches, each employing twelve participants who spent 100 min each learning 100 English words made up of 39 phonemes. Results in terms of the total number of words learned show that performance levels vary greatly among the individuals tested (with the best learners in both methods achieving word-recognition scores > 90%-correct on a 100-word vocabulary), both approaches are feasible for successful acquisition of word through the skin, and the phoneme-based approach provides a more consistent path for learning across users in a shorter period of time.

Abstract 3202: Universal detection of protein phosphorylation based on multi-functionalized soluble nanopolymers

Protein phosphorylation, as the most common covalent modification of proteins, plays a critical role in the regulation of many cellular functions. Irregularities in phosphorylation network are a major cause of onset and progression of many diseases, most notably cancer. Consequently, detection of protein phosphorylation is essential in further understanding of cellular signaling pathways and mechanisms of cell growth and proliferation. To assist in effective phosphorylation analyses, we introduce here a novel technique based on soluble nanopolymers with excellent solubility, compact spherical shape and chemical homogeneity. Soluble nanopolymers functionalized with Ti(IV) ions have shown highly versatility and allow for highly efficient analyses of phosphorylation and signaling pathways. We devise here a novel strategy, termed pIMAGO (phospho-imaging), for effective detection of phosphoproteins on a membrane (Western Blot format) or in a 96-well plate (ELISA format). Unlike antibody-based strategies, the new method is capable of selectively binding a phosphorylated residue independent of amino acid microenvironment. It shows no preference for either of the phosphosites, and thus holds great promise in biological analyses where the site of phosphorylation is not known or its specific antibody is not available. Using this technique, the phosphorylation levels of proteins of interest under physiological conditions can be readily detected as part of a standard Western Blot or ELISA procedures without the need for radioactivity or expensive phosphosite-specific antibodies. The utility of the approach has been demonstrated using standard mixtures of proteins, by in vitro kinase and phosphatase assays, as well as with kinase inhibitor screening and enzyme profiling. In addition, multiplexing capabilities of pIMAGO are presented, a feature often unavailable during antibodies-based detection. Finally, we further confirmed that the technique is sensitive and specific enough to detect endogenous phosphorylation changes by analyzing physiological signaling in number of endogenous protein complexes isolated directly from cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3202. doi:1538-7445.AM2012-3202