Recently, we demonstrated that two members of neurotrophins, nerve growth factor and brain-derived neurotrophic factor, and two types of receptor, tyrosine kinase A (TrkA) and tyrosine kinase (TrkB), exist in ejaculated bull spermatozoa, and play a crucial role in the normal function of spermatozoa. Neurotrophin-4 (NT-4) is another neurotrophic factor ...

In the mammalian sperm, the acrosome reaction is considered to be a regulated secretion that is an essential requirement for physiological fertilization. The acrosome reaction is the all-or-nothing secretion system that allows for multiple membrane fusion events. It is a Ca2+-regulated exocytosis reaction that has also been shown to be ...

During the capacitation process, spermatozoa acquire the ability to fertilize an oocyte, and up regulation of cAMP-dependent protein tyrosine phosphorylation occurs. Recently, Src family tyrosine kinase (SFK) has been involved in spermatozoa capacitation as a key PKA-dependent tyrosine kinase in several species. This work investigates the expression and role of ...

Protein tyrosine phosphorylation in sperm is associated with capacitation in several mammalian species. Although tyrosine phosphorylated proteins have been demonstrated in cryopreserved sperm, indicating capacitation-like changes during cryopreservation, these changes have not yet been quantified objectively. We monitored tyrosine phosphorylation, intracellular calcium and sperm kinematics throughout the cryopreservation process, and ...

We studied the effect of hypoxia on the differentiation of chronic myeloid leukemic K562 cells, and characterized the role of Na+/H+ exchanger 1 (NHE1) in this process. The K562 cells were tested for differentiation-related morphological characterization using light microscopy. The fluorescent probe BCECF was used for intracellular pH (pHi) measurements. ...

Fertilization is the process by which male and female haploid gametes (sperm and egg) unite to produce a genetically distinct individual. In mammals, fertilization involves a number of sequential steps, including sperm migration through the female genital tract, sperm penetration through the cumulus mass, sperm adhesion and binding to the ...

Primary cilia are microtubule-based solitary membrane projections on the cell surface that play important roles in signaling and development. Recent studies have demonstrated that polarized vesicular trafficking involving the small GTPase Rab8 and its guanine nucleotide-exchange factor Rabin8 is essential for primary ciliogenesis. In this study, we reveal that a ...

The neural cell adhesion molecule (NCAM) plays a fundamental role during development and regeneration. NCAM is expressed in three major isoforms, two of them with intracellular domains of different length and one without any intracellular domain. The cytoplasmic domain of NCAM contains, depending on the isoform, up to 49 phosphorylation ...

The present study was conducted to know the role of Nitric Oxide (NO) on the acrosome reaction (AR) in Murrah buffalo (Bubalus bubalis) spermatozoa. Ejaculated buffalo spermatozoa were washed, suspended in sp-TALP media containing 6mg BSA/mL and cell concentration was adjusted to 50×10(6)cells/mL. The cells were incubated for 6h in ...

Ras-like (Ral) small GTPases are regulated downstream of Ras and the noncanonical Ral guanine nucleotide exchange factor (RalGEF) effector pathway. Despite RalA and RalB sharing 82% sequence identity and utilization of shared effector proteins, their roles in normal and neoplastic cell growth have been shown to be highly distinct. Here ...

NEDD9, a member of the Cas family of signal transduction molecules, is amplified at the genetic level in melanoma and elevated expression levels have been shown to correlate with melanoma progression and metastasis. While NEDD9 interacts with the guanine nucleotide exchange factor DOCK3 to promote Rac activation and the elongated, ...

Response regulators (RRs) belong to two-component signaling pathways, widely prevalent in bacteria and lower eukaryotes, for sensing and mediating responses to diverse environmental stress stimuli. RRs are modular proteins, and in most instances, a receiver domain is found connected to diverse effector domain(s). All receiver domains contain a conserved aspartate, ...

Many stimuli mediate activation and nuclear translocation of ERK (extracellular-signal-regulated kinase) by phosphorylation on the TEY (Thr-Glu-Tyr) motif. This is necessary to initiate transcriptional programmes controlling cellular responses, but the mechanisms that govern ERK nuclear targeting are unclear. Single-cell imaging approaches have done much to increase our understanding of input-output ...

BCL2L12 has been reported to be involved in post-mitochondrial apoptotic events in glioblastoma, but the role of BCL2L12A, a splicing variant of BCL2L12, remains unknown. In this study, we showed that BCL2L12 and BCL2L12A were overexpressed in glioblastoma multiforme (GBM). Large-scale yeast two-hybrid screening showed that BCL2L12 was a GSK3b ...

Detection of activated caspases of spermatozoa could be helpful to evaluate male infertility. Although western blot is validated as a highly specific method to detect the proteins extracted from cells, the ability of this technique to detect activated sperm caspases in human semen may be limited. Indeed, round cells, which ...

Many motile processes are regulated such that movement occurs only upon activation of a signaling cascade. Sperm from a variety of species are initially quiescent and must be activated prior to beating. The signaling events leading to the activation and regulation of sperm motility are not well characterized. Mature seminal ...

Protein serine-threonine kinase casein kinase II (CK2) is involved in a myriad of cellular processes including cell growth and proliferation through its phosphorylation of hundreds of substrates, yet how CK2 function is regulated is poorly understood. Here we report that the CK2 catalytic subunit CK2α is modified by O-linked β-N-acetyl-glucosamine ...

Abstract. Using a newly developed competitive binding assay dependent upon the reassembly of a split reporter protein, we have tested the promiscuity of a panel of reported kinase inhibitors against the AGC group. Many non-AGC targeted kinase inhibitors target multiple members of the AGC group. In general, structurally similar inhibitors ...

ATR kinase activates the S-phase checkpoint when replication forks stall at sites of DNA damage. This event also causes phosphorylation of the Fanconi anemia (FA) protein FANCI, triggering its monoubiquitination of the key DNA repair factor FANCD2 by the FA core E3 ligase complex, thereby promoting this central pathway of ...

P‑Rex1 is a guanine-nucleotide exchange factor (GEF) for the small G protein Rac that is activated by PIP3 and Gbg subunits and inhibited by PKA. Here, we show that Protein Phosphatase 1a (PP1a) binds P‑Rex1 through an RVxF-type docking motif. PP1a activates P‑Rex1 directly in vitro, both independently of and ...

Most protein kinases share a DFG (Asp-Phe-Gly) motif in the ATP site which can assume two distinct conformations, the active DFG-in and the inactive DFG-out states. Small molecule inhibitors able to induce the DFG-out state have received considerable attention in kinase drug discovery. Using a typical DFG-in inhibitor scaffold of ...

In our continuing search for medicinal agents to treat proliferative diseases, quinazoline derivatives were synthesized and evaluated pharmacologically as EGFR and VEGFR-2 tyrosine kinase inhibitor. A QSAR analysis was conducted in order to rationalize the structure-activity relationship and to predict how similar the inhibitor-binding profiles of two protein kinases are ...

The canonical pathway of regulation of the germinal centre kinase (GCK) III subgroup member, mammalian Sterile20-related kinase 3 (MST3), involves a caspase-mediated cleavage between N-terminal catalytic and C-terminal regulatory domains with possible concurrent autophosphorylation of the activation loop MST3(Thr178-), induction of Ser-/Thr-protein kinase activity and nuclear localisation. We identified an ...

In this study, an integrated approach is developed for the formation, identification and biological characterization of electrochemical conversion products of p38α mitogen-activated protein kinase inhibitors. This work demonstrates the hyphenation of an electrochemical reaction cell with a continuous-flow bioaffinity assay and parallel LC-HR-MS. Competition of the formed products with a ...

PICK1 (protein interacting with C kinase 1) contains an N-terminal protein binding PDZ domain and a C-terminal lipid binding BAR domain. PICK1 plays a key role in several physiological processes, including synaptic plasticity. However, little is known about the cellular mechanisms governing the activity of PICK1 itself. Here we show ...

Eukaryotic elongation factor 2 kinase (eEF2K) phosphorylates and inactivates the translation elongation factor eEF2. eEF2K is not a member of the main eukaryotic protein kinase superfamily but instead belongs to a small group of so-called a-kinases. The activity of eEF2K is normally dependent upon Ca2+ and calmodulin. eEF2K has previously ...

The mTOR complex 2 (mTORC2) is a protein kinase complex involved in many important physiological processes through the regulation of its substrates, such as Akt, SGK1, and conventional PKC. Its activity is modulated negatively by interaction with DEPTOR and positively by the TSC1-TSC2 protein complex. To study the regulation of ...

Natural killer (NK) cells contribute to the defense against infected and transformed cells through the engagement of multiple germline-encoded activation receptors. Stimulation of the Fc receptor CD16 alone is sufficient for NK cell activation, whereas other receptors, such as 2B4 (CD244) and DNAM-1 (CD226), act synergistically. After receptor engagement, protein ...

We have previously characterized the interactions of the response regulator ComE from Streptococcus mutans and DNA binding sites through DNaseI footprinting and electrophoretic mobility shift assay analysis. Since response regulator functions are often affected by their phosphorylation state, we investigated how phosphorylation affects the biochemical function of ComE. Unlike many ...

The decision between survival and death in cells exposed to TNF relies on a highly regulated equilibrium between proapoptotic and antiapoptotic factors. The TNF-activated antiapoptotic response depends on several transcription factors, including NF-κB and its RelA/p65 subunit, that are activated through phosphorylation-mediated degradation of IκB inhibitors, a process controlled by ...

Transient receptor potential melastatin 7 (TRPM7) channels are divalent cation-selective ion channels that are permeable to Ca(2+) and Mg(2+). TRPM7 is ubiquitously expressed in vertebrate cells and contains both an ion channel and a kinase domain. TRPM7 plays an important role in regulating cellular homeostatic levels of Ca(2+) and Mg(2+) ...

In this report, we describe the phosphorylation of Yin Yang 1 (YY1) in vitro and in vivo by CK2α (Casein Kinase II), a multifunctional serine/threonine protein kinase. YY1 is a ubiquitously expressed multifunctional zinc finger transcription factor implicated in regulation of many cellular and viral genes. The products of these ...

Osteopontin (OPN) is a multifunctional phosphorylated protein containing the integrin binding sequence Arg-Gly-Asp through which it interacts with several integrin receptors, such as the αVβ3-integrin. OPN exists in many different isoforms differing in phosphorylation status that are likely to interact differently with integrins. The C-terminal region of OPN is particularly ...

The mammalian target of rapamycin (mTOR) is an evolutionarily conserved protein kinase that belongs to the phosphatidylinositol kinase-related kinase family. We describe our molecular characterization of mTOR and its function (GenBank accession HM114224) in Cashmere goat (Capra hircus). The goat mTOR complementary DNA is 8617 bp, comprising an open reading frame ...

Many recent high throughput technologies have enabled large-scale discoveries of new phosphorylation sites and phosphoproteins. Although they have provided a number of insights into protein phosphorylation and the related processes, an inclusive analysis on the nature of phosphorylated sites in proteins is currently lacking. We have therefore analyzed the occurrence ...

Cbl-b is a RING-type E3 ubiquitin ligase that functions as a negative regulator of T-cell activation and growth factor receptor and nonreceptor-type tyrosine kinase signaling. Cbl-b dysfunction is related to autoimmune diseases and cancers in humans. However, the molecular mechanism regulating its E3 activity is largely unknown. NMR and small-angle ...

Posttranslational modifications offer a dynamic way to regulate protein activity, subcellular localization, and stability. Here we estimate the effect of phosphorylation on protein binding and function for different types of complexes from human proteome. We find that phosphorylation sites tend to be located on binding interfaces in heterooligomeric and weak ...

FOXO3a is a forkhead transcription factor that regulates a multitude of important cellular processes, including proliferation, apoptosis, differentiation and metabolism. Doxorubicin treatment of MCF-7 breast carcinoma cells results in FOXO3a nuclear relocation and the induction of the stress-activated kinase p38-MAPK. In here, we studied the potential regulation of FOXO3a by ...

Eukaryotic elongation factor 2 kinase (eEF2K) is a calcium-calmodulin dependent protein kinase which regulates the translation elongation machinery. eEF2K belongs to the small group of so-called 'a-kinases' which are distinct from the main eukaryotic protein kinase superfamily. In addition to the a-kinase catalytic domain, other domains have been identified in ...

Protein kinases are key mediators of cellular signaling, and therefore, their activities are tightly controlled. AGC kinases are regulated by phosphorylation and by N- and C-terminal regions. Here, we studied the molecular mechanism of inhibition of atypical PKCζ and found that the inhibition by the N-terminal region cannot be explained ...

Phosphorylation of the BH3-only protein BAD can either directly disrupt its association with pro-survival proteins BCL-XL and/or BCL-2, or cause association of BAD with 14-3-3 proteins. Here we further characterize phosphorylation of BAD at Ser170, a unique with unclear function. We provide further evidence that mutation of Ser170 to a ...

In this paper, we present the structure-based design, synthesis and biological activity of N-pyrazole, N'-thiazole-ureas as potent inhibitors of p38α mitogen-activated protein kinase (p38α MAPK). Guided by complex crystal structures, we employed the initially identified N-aryl, N'-thiazole urea scaffold and introduced key structural elements that allowed the formation of novel ...

Protein kinases are activated by phosphorylation and by the binding of activator proteins. The interplay of these two factors is incompletely understood. We have applied energetic analysis to this question and have characterized the activation process of the serine/threonine kinase Aurora-A by phosphorylation and by its protein partner TPX21. We ...

The tripartite motif (TRIM)-containing proteins are a family of proteins that have been known to be involved in divergent biological processes, including important roles in immune responses through regulating various signaling pathways. In this study, we identified a member of the TRIM family, TRIM8, as a positive regulator of tumor ...

Autophagic responses are coupled to the activation of the inhibitor of NF-κB kinase (IKK). Here, we report that the essential autophagy mediator Beclin 1 and TGFβ-activated kinase 1 (TAK1)-binding proteins 2 and 3 (TAB2 and TAB3), two upstream activators of the TAK1-IKK signalling axis, constitutively interact with each other via ...

CD84 is a self-binding receptor from the CD150 (or signaling lymphocyte activation molecule [SLAM]) family that is broadly expressed in hematopoietic cells. It has been described that the adaptors SLAM-associated protein (SAP) and EWS-FLI1-activated transcript 2 (EAT-2) are critical for CD150 family members' signaling and function. We observed that human ...

Arrestins are a small family of proteins with four mammalian members that play key roles in the regulation of multiple GPCR-dependent and -independent signaling pathways. Although arrestins were reported to serve as scaffolds for MAP kinase cascades, promoting the activation of JNK3, ERK1/2, and p38, the molecular mechanisms involved were ...

Protein kinases are key signaling enzymes that catalyze the transfer of γ-phosphate from an ATP molecule to a phospho-accepting residue in the substrate. Unraveling the molecular features that govern the preference of kinases for particular residues flanking the phosphoacceptor is important for understanding kinase specificities toward their substrates and for ...

Activation of pro-inflammatory transcription factors NF-κB and signal transducer and activator of transcription 3 (STAT3) is one of the major contributors to both pathogenesis and chemoresistance in multiple myeloma (MM), which results in high mortality rate. Thus, in the present study, we investigated whether celastrol could suppress the proliferation and ...

The cytoskeletal adaptor protein paxillin localizes to the microtubule organizing center (MTOC) in T cells and, upon target cell binding, is recruited to the supramolecular activation complex (SMAC). We mapped the region of paxillin that associates with both the MTOC and SMAC to the leucine-aspartic acid (LD) domains and showed ...