Bisphenol S Reduces Pig Spermatozoa Motility through Different Intracellular Pathways and Mechanisms than Its Analog Bisphenol A.

Bisphenol A (BPA: 2,3-bis (4-hydroxyphenyl) propane) is an environmental chemical widely used in the manufacturing of epoxy polymers and many thermoplastic consumer products. Serious concerns about its safety led to the development of analogs, such as BPS (4-hydroxyphenyl sulfone). Very limited studies about BPS's impact on reproduction, specifically in spermatozoa, exist in comparison with BPA. Therefore, this work aims to study the in vitro impact of BPS in pig spermatozoa in comparison with BPA, focusing on sperm motility, intracellular signaling pathways and functional sperm parameters. We have used porcine spermatozoa as an optimal and validated in vitro cell model to investigate sperm toxicity. Pig spermatozoa were exposed to 1 and 100 µM BPS or BPA for 3 and 20 h. Both bisphenol S and A (100 µM) significantly reduce pig sperm motility in a time-dependent manner, although BPS exerts a lower and slower effect than BPA. Moreover, BPS (100 µM, 20 h) causes a significant increase in the mitochondrial reactive species, whereas it does not affect sperm viability, mitochondrial membrane potential, cell reactive oxygen species, GSK3α/ß phosphorylation or phosphorylation of PKA substrates. However, BPA (100 µM, 20 h) leads to a decrease in sperm viability, mitochondrial membrane potential, GSK3ß phosphorylation and PKA phosphorylation, also causing an increase in cell reactive oxygen species and mitochondrial reactive species. These intracellular effects and signaling pathways inhibited might contribute to explaining the BPA-triggered reduction in pig sperm motility. However, the intracellular pathways and mechanisms triggered by BPS are different, and the BPS-caused reduction in motility can be only partially attributed to an increase in mitochondrial oxidant species.

Molecular Mechanisms Involved in the Impairment of Boar Sperm Motility by Peroxynitrite-Induced Nitrosative Stress.

Excessive levels of reactive nitrogen species (RNS) produce nitrosative stress. Among RNS is peroxynitrite, a highly reactive free radical generated when nitric oxide reacts with superoxide anion. Peroxynitrite effects have been mainly studied in somatic cells, and in spermatozoa the majority of studies are focused in humans. The aim of this study is to investigate the in vitro peroxynitrite effect on boar spermatozoa functions and the molecular mechanisms involved. Spermatozoa were exposed to the donor 3-morpholinosydnonimine (SIN-1) in non-capacitating or capacitating medium, motility was evaluated by CASA, functional parameters by flow cytometry and sperm protein phosphorylation by Western blotting. SIN-1 treatment, that significantly increases peroxynitrite levels in boar spermatozoa, potentiates the capacitating-stimulated phosphorylation of cAMP-dependent protein kinase 1 (PKA) substrates and GSK-3α. SIN-1 induced peroxynitrite does not decrease sperm viability, but significantly reduces sperm motility, progressive motility, velocities and motility coefficients. Concomitantly, peroxynitrite does not affect mitochondrial membrane potential, plasma membrane fluidity, or A23187-induced acrosome reaction. However, peroxynitrite significantly increases sperm lipid peroxidation in both media. In conclusion, peroxynitrite compromises boar sperm motility without affecting mitochondrial activity. Although peroxynitrite potentiates the phosphorylation of pathways leading to sperm motility, it also causes oxidative stress that might explain, at least partially, the motility impairment.

The calcium-sensing receptor regulates protein tyrosine phosphorylation through PDK1 in boar spermatozoa.

Regulation of protein tyrosine phosphorylation is required for sperm capacitation and oocyte fertilization. The objective of the present work was to study the role of the calcium-sensing receptor (CaSR) on protein tyrosine phosphorylation in boar spermatozoa under capacitating conditions. To do this, boar spermatozoa were incubated in Tyrode's complete medium for 4 hr and the specific inhibitor of the CaSR, NPS2143, was used. Also, to study the possible mechanism(s) by which this receptor exerts its function, spermatozoa were incubated in the presence of specific inhibitors of the 3-phosphoinositide dependent protein kinase 1 (PDK1) and protein kinase A (PKA). Treatment with NPS2143, GSK2334470, an inhibitor of PDK1 and H-89, an inhibitor of PKA separately induced an increase in tyrosine phosphorylation of 18 and 32 kDa proteins, a decrease in the serine/threonine phosphorylation of the PKA substrates together with a drop in sperm motility and viability. The present work proposes a new signalling pathway of the CaSR, mediated by PDK1 and PKA in boar spermatozoa under capacitating conditions. Our results show that the inhibition of the CaSR induces the inhibition of PDK1 that blocks PKA activity resulting in a rise in tyrosine phosphorylation of p18 and p32 proteins. This novel signalling pathway has not been described before and could be crucial to understand boar sperm capacitation within the female reproductive tract.

Boar sperm hyperactivated motility is induced by temperature via an intracellular calcium-dependent pathway.

Herein we describe a new protocol to induce boar sperm hypermotility: temperature-induced hypermotility (TIH). Briefly, spermatozoa stored at 17°C in a calcium-free Tyrode's basal medium (containing EGTA) were exposed to increased temperature by incubation at 38.5°C. Hypermotility induced by the calcium ionophore A23187 was used as a control (calcium ionophore-induced hyperactivity (CIIH)). The increase in temperature led to an increase in the percentage of hypermotile spermatozoa. When the slope of the temperature increase is near zero, sperm hyperactivity becomes a more progressive movement. Motility parameters of sperm hyperactivation induced by TIH were different from those following CIIH. Cluster analysis revealed that these two populations of hyperactivated spermatozoa are different. TIH is independent of extracellular Ca2+ but dependent on intracellular Ca2+ release. Moreover, TIH is unaffected by protein kinase A (PKA) inhibition, whereas CIIH is reduced by half in the presence of a PKA inhibitor. In conclusion, we have demonstrated that: (1) a temperature increase in boar spermatozoa is a stimulus that can induce a hyperactive population, which is differs from the hyperactive sperm population induced by calcium ionophore; (2) the temperature increase in spermatozoa triggers the release of Ca2+ from intracellular stores; (3) extracellular calcium is not required for TIH; and (4) TIH in boar spermatozoa is independent of PKA activity.

Src family tyrosine kinase regulates acrosome reaction but not motility in porcine spermatozoa.

During the capacitation process, spermatozoa acquire the ability to fertilize an oocyte, and upregulation 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 SFK in porcine spermatozoa. SFK members Lyn and Yes are identified in porcine spermatozoa by western blotting as well as two proteins named SFK1 and SFK2 were also detected by their tyrosine 416 phosphorylation, a key residue for SFK activation. Spermatozoa with SFK1 and SFK2 increase their Y416 phosphorylation time-dependently under capacitating conditions compared with noncapacitating conditions. The specific SFK inhibitor SU6656 unaffected porcine spermatozoa motility or viability. Moreover, SFK inhibition in spermatozoa under capacitating conditions leads to a twofold increase in both nonstimulated and calcium-induced acrosome reaction. Our data show that capacitating conditions lead to a time-dependent increase in actin polymerization in boar spermatozoa and that long-term incubation with SFK inhibitor causes a reduction in the F-actin content. In summary, this work shows that the SFK members Lyn and Yes are expressed in porcine spermatozoa and that SFK1 and SFK2 are phosphorylated (activated) during capacitation. Our results point out the important role exerted by SFK in the acrosome reaction, likely mediated in part by its involvement in the actin polymerization process that accompanies capacitation, and rule out its involvement in porcine spermatozoa motility.

Sperm Phosphoproteome: Unraveling Male Infertility.

Infertility affects approximately 15% of couples worldwide of childbearing age, and in many cases the etiology of male infertility is unknown. The current standard evaluation of semen is insufficient to establish an accurate diagnosis. Proteomics techniques, such as phosphoproteomics, applied in this field are a powerful tool to understand the mechanisms that regulate sperm functions such as motility, which is essential for successful fertilization. Among the post-translational modifications of sperm proteins, this review summarizes, from a proteomic perspective, the updated knowledge of protein phosphorylation, in human spermatozoa, as a relevant molecular mechanism involved in the regulation of sperm physiology. Specifically, the role of sperm protein phosphorylation in motility and, consequently, in sperm quality is highlighted. Additionally, through the analysis of published comparative phosphoproteomic studies, some candidate human sperm phosphoproteins associated with low sperm motility are proposed. Despite the remarkable advances in phosphoproteomics technologies, the relatively low number of studies performed in human spermatozoa suggests that phosphoproteomics has not been applied to its full potential in studying male infertility yet. Therefore, further studies will improve the application of this procedure and overcome the limitations, increasing the understanding of regulatory mechanisms underlying protein phosphorylation in sperm motility and, consequently, in male fertility.

Impaired mammalian sperm function and lower phosphorylation signaling caused by the herbicide Roundup® Ultra Plus are due to its surfactant component.

The use of worldwide glyphosate-based herbicide Roundup® is growing and to date its effects on mammalian spermatozoa are controversial. This study aims to investigate the functional impact of in vitro exposure of pig spermatozoa to low concentrations of Roundup® Ultra Plus (RUP), similar to those present as environment contaminants, to its active ingredient glyphosate, and to the non-active component, surfactant polyoxyethyleneamine (POEA). Pig spermatozoa were incubated in Tyrode's basal medium (TBM) or Tyrode's complete medium (TCM) (1 h at 38.5 °C) with several RUP dilutions or equivalent concentrations of glyphosate or POEA. RUP treatment causes a significant dilution-dependent decrease in sperm motility, a significant increase in plasma membrane disorganization and reduction in GSK3ß phosphorylation (TBM) and in two PKA substrates (TBM and TCM), whereas does not affect sperm viability or mitochondrial membrane potential (MMP). Equivalent glyphosate concentrations do not affect any functional sperm parameters. However, POEA concentrations equivalent to RUP dilutions mimic all RUP sperm effects: decrease sperm motility in a concentration-dependent manner, increase sperm plasma membrane lipid disorder and significantly inhibit GSK3ß phosphorylation (TBM) and two PKA substrates without affecting sperm viability or MMP. In summary, low concentrations RUP herbicide cause sperm motility impairment without affecting sperm viability. This adverse effect could be likely due to a detrimental effect in the plasma membrane lipid organization and to inhibition of phosphorylation of both, GSK3ß and specific PKA substrates. Importantly, our results indicate that negative effects of low RUP concentrations in pig spermatozoa function are likely caused by the surfactant included in its formulation and no by its active ingredient glyphosate.

Protein kinases A and C and phosphatidylinositol 3 kinase regulate glycogen synthase kinase-3A serine 21 phosphorylation in boar spermatozoa.

The cAMP-dependent protein kinase (PKA), protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K) pathways control most relevant functions in male germ cells including motility. Recently we demonstrated that phosphorylation state of glycogen synthase kinase-3alpha (GSK3A) is also a key event in the control of boar spermatozoa motility. However, the upstream regulators of GSK3A serine phosphorylation (inhibition) in male germ cells remain largely unknown. This work investigates the involvement of PKA, PKC and PI3K pathways in GSK3A phosphorylation in boar spermatozoa. A capacitating medium (TCM) or the phosphodiesterase-resistant cell permeable cAMP analogue 8Br-cAMP cause a significant increase in Ser21 GSK3A phosphorylation associated with a simultaneous significant increase in boar spermatozoa motility. These effects are blocked after preincubation of spermatozoa with PKA inhibitor H89 or PKC inhibitor Ro-32-0432. The PI3K inhibitor LY294002 increases both spermatozoa motility parameters and the basal GSK3A phosphorylation, but does not affect either TCM- or 8Br-cAMP-stimulated GSK3A phosphorylation. PI3K inhibition effects are mediated by an increase in intracellular cAMP levels in boar spermatozoa and are suppressed by PKA inhibitor H89. In summary, we demonstrate that PKA, PKC and PI3K pathways crosstalk in porcine male germ cells to crucially regulate GSK3A phosphorylation which subsequently controls cell motility. In addition, our results suggest that PI3K is upstream of PKA which lies upstream of PKC in this regulatory cascade(s). Our findings contribute to elucidate the molecular mechanisms underlying the regulation of one of the most relevant male germ cell functions, motility.

Study of the Metabolomics of Equine Preovulatory Follicular Fluid: A Way to Improve Current In Vitro Maturation Media.

Production of equine embryos in vitro is currently a commercial technique and a reliable way of obtaining offspring. In order to produce those embryos, immature oocytes are retrieved from postmortem ovaries or live mares by ovum pick-up (OPU), matured in vitro (IVM), fertilized by intracytoplasmic sperm injection (ICSI), and cultured until day 8-10 of development. However, at best, roughly 10% of the oocytes matured in vitro and followed by ICSI end up in successful pregnancy and foaling, and this could be due to suboptimal IVM conditions. Hence, in the present work, we aimed to elucidate the major metabolites present in equine preovulatory follicular fluid (FF) obtained from postmortem mares using proton nuclear magnetic resonance spectroscopy (1H-NMR). The results were contrasted against the composition of the most commonly used media for equine oocyte IVM: tissue culture medium 199 (TCM-199) and Dulbecco's modified eagle medium/nutrient mixture F-12 Ham (DMEM/F-12). Twenty-two metabolites were identified in equine FF; among these, nine of them are not included in the composition of DMEM/F-12 or TCM-199 media, including (mean ± SEM): acetylcarnitine (0.37 ± 0.2 mM), carnitine (0.09 ± 0.01 mM), citrate (0.4 ± 0.04 mM), creatine (0.36 ± 0.14 mM), creatine phosphate (0.36 ± 0.05 mM), fumarate (0.05 ± 0.007 mM), glucose-1-phosphate (6.9 ± 0.4 mM), histamine (0.25 ± 0.01 mM), or lactate (27.3 ± 2.2 mM). Besides, the mean concentration of core metabolites such as glucose varied (4.3 mM in FF vs. 5.55 mM in TCM-199 vs. 17.5 mM in DMEM/F-12). Hence, our data suggest that the currently used media for equine oocyte IVM can be further improved.

AMP-activated kinase in human spermatozoa: identification, intracellular localization, and key function in the regulation of sperm motility.

AMP-activated kinase (AMPK), a protein that regulates energy balance and metabolism, has recently been identified in boar spermatozoa where regulates key functional sperm processes essential for fertilization. This work's aims are AMPK identification, intracellular localization, and their role in human spermatozoa function. Semen was obtained from healthy human donors. Sperm AMPK and phospho-Thr172-AMPK were analyzed by Western blotting and indirect immunofluorescence. High- and low-quality sperm populations were separated by a 40%-80% density gradient. Human spermatozoa motility was evaluated by an Integrated Semen Analysis System (ISAS) in the presence or absence of the AMPK inhibitor compound C (CC). AMPK is localized along the human spermatozoa, at the entire acrosome, midpiece and tail with variable intensity, whereas its active form, phospho-Thr172-AMPK, shows a prominent staining at the acrosome and sperm tail with a weaker staining in the midpiece and the postacrosomal region. Interestingly, spermatozoa bearing an excess residual cytoplasm show strong AMPK staining in this subcellular compartment. Both AMPK and phospho-Thr172-AMPK human spermatozoa contents exhibit important individual variations. Moreover, active AMPK is predominant in the high motility sperm population, where shows a stronger intensity compared with the low motility sperm population. Inhibition of AMPK activity in human spermatozoa by CC treatment leads to a significant reduction in any sperm motility parameter analyzed: percent of motile sperm, sperm velocities, progressivity, and other motility coefficients. This work identifies and points out AMPK as a new molecular mechanism involved in human spermatozoa motility. Further AMPK implications in the clinical efficiency of assisted reproduction and in other reproductive areas need to be studied.

Cholecystokinin-stimulated tyrosine phosphorylation of PKC-delta in pancreatic acinar cells is regulated bidirectionally by PKC activation.

PKC-delta is important in cell growth, apoptosis, and secretion. Recent studies show its stability is regulated by tyrosine phosphorylation (TYR-P), which can be stimulated by a number of agents. Many of these stimuli also activate phospholipase C (PLC) cascades and little is known about the relationship between these cascades and PKC-delta TYR-P. Cholecystokinin (CCK) stimulates PKCs but it is unknown if it causes PKC-delta TYR-P and if so, the relationship between these cascades is unknown. In rat pancreatic acini, CCK-8 stimulated rapid PKC-delta TYR-P by activation of the low affinity CCK(A) receptor state. TPA had a similar effect. BAPTA did not decrease CCK-stimulated PKC-delta TYR-P but instead, increased it. A23187 did not stimulate PKC-delta TYR-P. Wortmannin and LY 294002 did not alter CCK-stimulated PKC-delta TYR-P. GF 109203X, at low concentrations, increased PKC-delta TYR-P stimulated by CCK or TPA and at higher concentrations, inhibited it. The cPKC inhibitors, Gö 6976 and safingol, caused a similar increase in TPA- and CCK-stimulated PKC-delta TYR-P. These results demonstrate that CCK(A) receptor activation causes PKC-delta TYR-P through activation of only one of its two receptor affinity states. This PKC-delta TYR-P is not directly influenced by changes in [Ca(2+)](i); however, the resultant activation of PKC-alpha has an inhibitory effect. Therefore, CCK activates both stimulatory and inhibitory PKC cascades regulating PKC-delta TYR-P and, hence, likely plays an important role in regulating PKC-delta degradation and cellular abundance.

The cholecystokinin system in the rat retina: receptor expression and in vivo activation of tyrosine phosphorylation pathways.

High levels of endogenous cholecystokinin (CCK) are present in the rat retina and restricted to amacrine cells. Two types of CCK receptors exist but their expression and intracellular transduction pathways coupled in the rat retina are unknown. The aims of this study were to investigate CCK receptors expression in rat retina and to study downstream tyrosine phosphorylation pathways. For this purpose, total mRNA isolated from rat retina was subjected to RT-PCR analysis. Isolated rat retinas were incubated in presence of CCK. Soluble proteins in retinal homogenates were immunoprecipitated with anti-phoshpotyrosine or anti-p130(Cas) specific monoclonal antibodies and subjected to SDS-PAGE, followed by Western blotting analysis. Both types of CCK receptor mRNAs, A and B, are present in the rat retina. Incubation of retina with CCK induced a rapid increase in several phosphotyrosine-containing bands with molecular masses greater than 30 kDa. Western Blotting and immunoprecipitation with a specific monoclonal antibody identified one of the phosphotyrosine bands as the adapter protein p130(Cas). Tyrosine phosphorylation of p130(Cas) induced by CCK in rat retina was time and concentration dependent: CCK induced tyrosine phosphorylation of p130(Cas) occurred rapidly with the maximum effect observed at 2.5 min incubation with 1 microM CCK. Our data clearly identified CCK-A and CCK-B receptor mRNAs in the rat retina and demonstrated that they are functional, stimulating tyrosine phosphorylation pathways. Our results provide novel biochemical information to further understand the physiological role of CCK A and B receptors in rat retina.

AMP-activated kinase AMPK is expressed in boar spermatozoa and regulates motility.

The main functions of spermatozoa required for fertilization are dependent on the energy status and metabolism. AMP-activated kinase, AMPK, acts a sensor and regulator of cell metabolism. As AMPK studies have been focused on somatic cells, our aim was to investigate the expression of AMPK protein in spermatozoa and its possible role in regulating motility. Spermatozoa from boar ejaculates were isolated and incubated under different conditions (38,5°C or 17°C, basal medium TBM or medium with Ca(2+) and bicarbonate TCM, time from 1-24 hours) in presence or absence of AMPK inhibitor, compound C (CC, 30 µM). Western blotting reveals that AMPK is expressed in boar spermatozoa at relatively higher levels than in somatic cells. AMPK phosphorylation (activation) in spermatozoa is temperature-dependent, as it is undetectable at semen preservation temperature (17°C) and increases at 38,5°C in a time-dependent manner. AMPK phosphorylation is independent of the presence of Ca(2+) and/or bicarbonate in the medium. We confirm that CC effectively blocks AMPK phosphorylation in boar spermatozoa. Analysis of spermatozoa motility by CASA shows that CC treatment either in TBM or in TCM causes a significant reduction of any spermatozoa motility parameter in a time-dependent manner. Thus, AMPK inhibition significantly decreases the percentages of motile and rapid spermatozoa, significantly reduces spermatozoa velocities VAP, VCL and affects other motility parameters and coefficients. CC treatment does not cause additional side effects in spermatozoa that might lead to a lower viability even at 24 h incubation. Our results show that AMPK is expressed in spermatozoa at high levels and is phosphorylated under physiological conditions. Moreover, our study suggests that AMPK regulates a relevant function of spermatozoa, motility, which is essential for their ultimate role of fertilization.

CCK1 and 2 receptors are expressed in immortalized rat brain neuroblasts: intracellular signals after cholecystokinin stimulation.

Cholecystokinin (CCK) is one of the most abundant neuropeptides in the central nervous system (CNS) where it promotes important functions by activation of receptors CCK1 and CCK2. Our aim was to investigate CCK receptors expression and their downstream intracellular signaling in immortalized rat brain neuroblasts. Results show that CCK1 and CCK2 receptor mRNAs and CCK2 receptor protein are expressed in neuroblasts. CCK incubation of neuroblasts leads to stimulation in a time-dependent manner of several signaling pathways, such as tyrosine phosphorylation of adaptor proteins paxillin and p130(Cas), phosphorylation of p44/p42 ERKs as well as PKB (Ser473). Moreover, CCK-8 stimulates the DNA-binding activity of the transcription factor AP-1. The CCK2 receptor agonist gastrin stimulates ERK1/2 phosphorylation in a comparable degree as CCK does. ERK1/2 phosphorylation activated by CCK-8 was markedly inhibited by the CCK2 receptor antagonist CR2945. Incubation for 48 h with CCK-8 increases neuroblasts viability in a similar degree as EGF. In summary, our data clearly identify CCK1 and CCK2 receptor mRNAs and CCK2 receptor protein in brain neuroblasts and show that incubation with CCK promotes cell proliferation and activates the phosphorylation of survival transduction pathways. Stimulation of ERK1/2 phosphorylation by CCK is mainly mediated by the CCK2 receptor. Moreover, this work might provide a novel model of proliferating neuronal cells to further study the biochemical mechanisms by which the neuropeptide CCK exerts its actions in the CNS.

Adapter protein CRKII signaling is involved in the rat pancreatic acini response to reactive oxygen species.

Recent studies demonstrate that reactive oxygen species (ROS) are important mediators of acute pancreatitis, whether induced experimentally or in necrotizing pancreatitis in humans; however, the cellular processes involved remain unclear. Adapter protein CrkII, plays a central role for convergence of cellular signals from different stimuli. Cholecystokinin (CCK), which induces pancreatitis, stimulates CrkII tyrosine phosphorylation and CrkII protein complexes, raising the possibility it can be important in the acinar cell responses to ROS. Therefore, our aim was to investigate whether CrkII signaling is involved in the biological response of rat pancreatic acini to H2O2 and the intracellular mediators implicated. Treatment of isolated rat pancreatic acini with H2O2 rapidly stimulates CrkII phosphorylation, measured as electrophoretic mobility shift and by using a phosphospecific antibody (pTyr221). Tyrosine kinase blocker B44 inhibits the higher phosphorylation state, demonstrating that it occurs mainly in tyrosine residues. H2O2-induced CrkII phosphorylation is time- and concentration-dependent, showing maximal effect with 3 mM H2O2 at 5 min. The intracellular pathways induced by H2O2 leading to CrkII tyrosine phosphorylation do not involve PKC, intracellular calcium, PI3-K or the actin cytoskeleton integrity. ROS generation clearly promotes the formation of protein complex CrkII-PYK2. In conclusion, ROS clearly affect the key adapter protein CrkII signaling by two ways: stimulation of CkII phosphorylation and a functional consequence: formation of CrkII-protein complexes. Because of its central role in activating more distal pathways, CrkII might likely play an important role in the ability of ROS to induce pancreatic cellular injury and pancreatitis.

Phosphospecific site tyrosine phosphorylation of p125FAK and proline-rich kinase 2 is differentially regulated by cholecystokinin receptor type A activation in pancreatic acini.

The focal adhesion kinases, p125FAK and proline-rich kinase 2 (PYK2), are involved in numerous processes as adhesion, cytoskeletal changes, and growth. These kinases have 45% homology and share three tyrosine phosphorylation (TyrP) sites. Little information exists on the ability of stimulants to cause TyrP of each kinase site and the cellular mechanism involved. We explored the ability of the neurotransmitter/hormone, CCK, to stimulate TyrP at each site. In rat pancreatic acini, CCK stimulated TyrP at each site in both kinases. TyrP was rapid except for pY397FAK. The magnitude of TyrP differed with the different FAK and PYK2 sites. The CCK dose-response curve for TyrP for sites in each kinase was similar. CCK-JMV, an agonist of the high affinity receptor state and antagonist of the low affinity receptor state, was less efficacious than CCK at each FAK/PYK2 site and inhibited CCK maximal stimulation. Thapsigargin decreased CCK-stimulated TyrP of pY402PYK2 and pY925FAK but not the other sites. GF109203X reduced TyrP of only the PYK2 sites, pY402 and pY580. GF109203X with thapsigargin decreased TyrP of pY402PYK2 and the three FAK sites more than either inhibitor alone. Basal TyrP of pY397FAK was greater than other sites. These results demonstrate that CCK stimulates tyrosine phosphorylation of each of the three homologous phosphorylation sites in FAK and PYK2. However, CCK-stimulated TyrP at these sites differs in kinetics, magnitude, and participation of the high/low affinity receptor states and by protein kinase C and [Ca2+]i. These results show that phosphorylation of these different sites is differentially regulated and involves different intracellular mechanisms in the same cell.

Cholecystokinin rapidly stimulates CrkII function in vivo in rat pancreatic acini. Formation of CrkII-protein complexes.

Crk belongs to a family of adapter proteins whose structure allows interaction with tyrosine-phosphorylated proteins and is therefore an important modulator of downstream signals, representing a convergence of the actions of numerous stimuli. Recently, it was demonstrated that cholecystokinin (CCK) induced tyrosine phosphorylation of proteins related to fiber stress formation in rat pancreatic acini. Here, we investigated whether CCK receptor activation signals through CrkII and forms complexes with tyrosine-phosphorylated proteins in rat pancreatic acini. We demonstrated that CCK promoted the transient formation of CrkII-paxillin and CrkII-p130Cas complexes with maximal effect at 1 min. Additionally, CCK decreased the electrophoretic mobility of CrkII. This decrease was time- and concentration-dependent and inversely related with its function. Carbachol and bombesin also decreased CrkII electrophoretic mobility, whereas epidermal growth factor, vasoactive intestinal peptide, secretin or pituitary adenylate cyclase-activating polypeptide had no effect. CCK-induced CrkII electrophoretic shift was dependent on the Src family of tyrosine kinases and occurred in the intact animal, suggesting a physiological role of CrkII mediating CCK actions in the exocrine pancreas in vivo.