Lawrence G Puente's research while affiliated with The Ottawa Hospital and other places

Publications (22)

Article
Full-text available
In complex organisms, caspase proteases mediate a variety of cell behaviors, including proliferation, differentiation, and programmed cell death/apoptosis. Structural homologs to the caspase family (termed metacaspases) engage apoptosis in single-cell eukaryotes, yet the molecular mechanisms that contribute to nondeath roles are currently undefined...
Article
Protein phosphorylation is a widespread cellular process, and simplistic linear pathway models of kinase signaling likely under-represent the complexity of in vivo pathways. The recent massive increase in information available through protein interaction databases now allows construction of in silico models of protein networks that are underpinned...
Data
DNA staining and flow cytometry gates. (a) 63× phase contrast/FITC microscope merge of sytox green stained yeast cells arrested with nocodazole. Microscopy was performed on an Axioplan 2 compound microscope fitted with a Zeiss Axiocam. Fixed cells were suspended in 50mM sodium citrate, slide mounted and viewed at 63× magnification with a PlanApochr...
Data
Expression of yca1 after cdc15 synchronization. Adaptation of data from Spellman et al. (Mol Biol Cell 9, 3273-97 1998) depicting expression of YCA1 after CDC15 synchronization. Arrows denote the first observation of new buds. Peak expression after 150 minutes release coincides with second appearance of small buds indicative of the G1/S transition...
Data
Expression of yca1 flanking genes. a) Region of chromosome XV depicting genes flanking yca1. b) Reverse transcriptase PCR of flanking genes demonstrates invariable expression in the Δyca1 and C297A strains when compared to the congenic BY4741 background strain. ADH3 was used as a loading control. (0.20 MB DOC)
Data
Verification of integration, deletion and expression. Integration of the C297A construct into the Δyca1 background strain. (a,b) The location of integration and expression of integrants were verified using A–B and C–D primers designed for the systematic deletion project (http://www-sequence.stanford.edu/group/yeast_deletion_project/confirmation.htm...
Article
Full-text available
Caspase proteases are a conserved protein family predominantly known for engaging and executing apoptotic cell death. Nevertheless, in higher eukaryotes, caspases also influence a variety of cell behaviors including differentiation, proliferation and growth control. S. cerevisiae expresses a primordial caspase, yca1, and exhibits apoptosis-like dea...
Article
Phosphorylation is one of the most abundant post-translational modifications on protein and one that frequently has functional biological consequences. For this reason, screening protein samples for phosphorylations has become an important tool in biochemical research. Affinity purification by immunological or chemical reagents can be used to isola...
Article
Full-text available
Multiple kinase activities are required for skeletal muscle differentiation. However, the mechanisms by which these kinase pathways converge to coordinate the myogenic process are unknown. Using multiple phosphoprotein and phosphopeptide enrichment techniques we obtained phosphopeptides from growing and differentiating C2C12 muscle cells and determ...
Article
Stimulation of cytotoxic T lymphocyte (CTL) degranulation with plate-bound anti-CD3 Ab leads to two phases of ERK activation: an early PKC-independent phase followed by a later sustained PKC-dependent phase. Herein, we show that a novel PKC (nPKC) mediates the late phase of ERK activation, upstream of Ras in murine T cells. In contrast, when CTL ar...
Article
Full-text available
Embryonic stem cells are a unique cell population capable both of self-renewal and of differentiation into all tissues in the adult organism. Despite the central importance of these cells, little information is available regarding the intracellular signaling pathways that govern self-renewal or early steps in the differentiation program. Embryonic...
Article
In this study, we examined the contribution of the actin cytoskeleton to T-cell receptor (TCR)-initiated signalling in cytotoxic T lymphocytes (CTLs). We demonstrate that cytoskeletal remodelling is required for sustaining TCR-stimulated signals that lead to degranulation by CTLs. Disruption of the actin cytoskeleton in CTLs already undergoing sign...
Article
Protein kinase C (PKC) theta plays a crucial role in T cell activation. We, therefore, examined the regulation of PKCtheta activity in cytotoxic T lymphocytes (CTL). We demonstrated that PMA did not stimulate PKCtheta activation and phospholipase C inhibition did not block anti-CD3-stimulated PKCtheta activation in a CTL clone. This suggests that d...
Article
The differentiation of skeletal muscle has been associated with altered phosphorylation status of individual proteins. However, a global analysis of protein phosphorylation during myogenesis has yet to be undertaken. Here, we report the identification of over 130 putative phosphoproteins from murine C2C12 muscle cells. Cell extracts were fractionat...
Article
beta 3 integrins mediate fibronectin binding and enhanced activation of cytotoxic T lymphocytes (CTL). The intracellular signals initiated by beta 3 integrins in lymphocytes are not well characterized, but in many cell types, beta 1 integrin ligation activates mitogen-activated protein (MAP) kinases. In the present study, we find that fibronectin c...
Article
β3 integrins mediate fibronectin binding and enhanced activation of cytotoxic T lymphocytes (CTL). The intracellular signals initiated by β3 integrins in lymphocytes are not well characterized, but in many cell types, β1 integrin ligation activates mitogen-activated protein (MAP) kinases. In the present study, we find that fibronectin can synergize...
Article
Activation of mitogen-activated protein kinases (MAPK) is a critical signal transduction event for CTL activation, but the signaling mechanisms responsible are not fully characterized. Protein kinase C (PKC) is thought to contribute to MAPK activation following TCR stimulation. We have found that dependence on PKC varies with the method used to sti...
Article
Requirements for T cell activation are not fully established. One model is that receptor occupancy and down-regulation are essential for activation, and another, not necessarily mutually exclusive, model is that sustained signals are important. Here we examine the importance of signal duration in T cell activation. First, we demonstrate that immobi...

Citations

... Our experimental model consisted of the Yca1 sequence fused to the mCherry tag (RFP), which was expressed from a plasmid under the control of the Adh1 promoter in the Δyca1 background 7 . This approach is an advance over our prior experimental model, as the constitutive expression of Yca1 in the current study is conducive for increased capture of protein interactions across all stoichiometries, which is an advance over our prior experimental model of defining Yca1 protein partners 7,21 . Proteins interacting with Yca1 were isolated by co-immunoprecipitation against the tag and identified using LC-MS/MS. ...
... Yca1 is important for life-span extension in yeast, where it plays a role in the management and removal of protein aggregates that would otherwise be toxic to cell survival (Lee et al., 2010;Shrestha et al., 2019). Interestingly, Yca1-deficient yeast cells not only accumulate misfolded protein aggregates (Lee et al., 2010;Shrestha et al., 2019), they also exhibit a delay in cell cycle progression (Lee et al., 2008) and a shorter life span (Hill et al., 2014), both activities found to be operative in multicellular organisms, including mammals as noted above. ...
... Phosphorylation of PIN hydrophilic loops is positively regulated by PID and negatively by PP2A activity [ 59 ]. Plant mutants of PP2A catalytic C subunits (C1-C5) indicate functions in ABA signaling (C2; [ 61 ]), brassinosteroid signaling (C5; [ 62 ]), and plant defense responses [ 63 ]; however related functions have also been described for PP2A-A and B subunits [ 62 , 64 , 65 ], including metabolism regulation [ 66 , 67 ]. Furthermore, PP2A negatively regulates accumulation and activity of ACS isozymes in ethylene production [ 68 ], as pp2a -a1 ( rcn1 ) mutant plants produce higher amounts of ethylene [ 69 ]. ...
Reference: The Plant Kinome
... Moreover, metacaspases act as monomers and require calcium for their activation, unlike caspases which form dimers prior to their molecular activation. Their substrate specificities are also different; metacaspases cleave peptides after arginine and lysine residues, but caspases cleave after aspartate residues (Lee et al., 2008). ...
... Comparably, CD4 + T cell polarization can be also controlled by signal intensity 26,27 . In addition, signal duration can also modulate T cell functionality as induction of transient signals corresponds with unresponsiveness or apoptosis, in contrast to sustained signaling, which results in activation and proliferation 10,11,28 . Finally, it has been postulated that the magnitude of activation signal is even important for T cell maturation 29 . ...
... It has been demonstrated that LPS induces proinflammatory cytokines through ERK1/2, JNK1/2 and p38 in macrophages [21,22]. In addition, LPS-mediated PKC activation induces the downstream signaling pathways such as MAPK [23,24] or NF-κB [25], that regulate the expression of IL-1β, IL-6,and TNF-α in macrophages. ...
... 36,39 A soluble mutated version of the RGD peptide, Cyclo-RAD-fluoromethylketone (RAD) was used as a control treatment. The CD29 (HUTS-21) antibody specifically ligates b1 integrin, 4,40 and it was injected intraocularly to investigate its effects on RGC survival. To verify this, YIGSR peptide, 41 which also binds to b1 integrin without inducing cell survival, was also injected intraocularly. ...
... The highly conserved acidic nuclear phosphoprotein 32 kDa (ANP32) family of proteins are characterized by an N-terminal leucine-rich repeat (LRR) domain and a C-terminal low-complexity acidic region (LCAR) (6). These proteins have been demonstrated to be involved in a wide array of physiological processes, including cell apoptosis (7)(8)(9)(10)(11)(12), differentiation (13)(14)(15)(16) and proliferation (17)(18)(19). The majority of studies on the ANP32 family have focused on ANP32A, which has been implicated in a variety of cellular functions. ...
... Earlier studies of PKC-θ phosphorylation in T cells used an electrophoretic mobility shift of PKC-θ in T cells upon anti-CD3/CD28 or PMA/ionomycin stimulation; these studies show that PKC-θ migrates more slowly in activated T cells than in unstimulated T cells (Thebault and Ochoa-Garay, 2004; Puente et al., 2005). Although the phosphorylation of PKC-θ at T538 is not directly examined in these studies due to the lack of use of anti-phospho-PKC-θ (T538) antibody, PKC-θ T538A mutation abolishes the mobility shift of PKC-θ induced by PMA (Thebault and Ochoa-Garay, 2004; Puente et al., 2005), suggesting that T538 phosphorylation is induced by T cell signaling. Using the phospho-specific PKC-θ T538 antibodies from different sources, several groups show that T538 phosphorylation is induced during T cell activation upon anti-CD3 or interferon stimulation (Srivastava et al., 2004; Lee et al., 2005; Liu and Lai, 2005; Park et al., 2009; Chuang et al., 2011). ...
... It is also of interest in this context that actin filament organization regulates cell effector functions. This is evident by treatment with cytochalasin, which inhibits actin polymerization and leads to insufficient T cell activation [58]. In non-immune cells, adenocarcinoma cells or lens epithelial cells, a pharmacological modification of actin-filament polymerization was sufficient to induce ion channel activity [59,60]. ...