Pyk2 Expression Research Articles

Tumor Necrosis Factor-α Stimulates Focal Adhesion Kinase Activity Required for Mitogen-activated Kinase-associated Interleukin 6 Expression

Tumor Necrosis Factor-α Stimulates Focal Adhesion Kinase Activity Required for Mitogen-activated Kinase-associated Interleukin 6 Expression

The Role of Proline-Rich Protein Tyrosine Kinase 2 in Differentiation-Dependent Signaling in Human Epidermal Keratinocytes

Non-receptor tyrosine kinase proline-rich protein tyrosine kinase 2 (Pyk2) functions as an integrator of multiple signaling pathways involved in the regulation of fundamental cellular processes. Pyk2 expression, regulation, and functions in skin have not been examined. Here we investigated the expression and subcellular localization of Pyk2 in human epidermis and in primary human keratinocytes, and studied the mechanisms of Pyk2 activation by differentiation-inducing stimuli, and the role of Pyk2 as a regulator of keratinocyte differentiation. We demonstrate that Pyk2 is abundantly expressed in skin keratinocytes. Notably, the endogenous Pyk2 protein is predominantly localized in keratinocyte nuclei throughout all layers of healthy human epidermis, and in cultured human keratinocytes. Pyk2 is activated by treatment with keratinocyte-differentiating agents, 12-O-tetradecanoylphorbol-13-acetate and calcium via a mechanism that requires intracellular calcium release and functional protein kinase C (PKC) and Src activities. Particularly, differentiation-promoting PKC delta and PKC eta elicit Pyk2 activation. Our data show that Pyk2 increases promoter activity and endogenous protein levels of involucrin, a marker of keratinocyte terminal differentiation. This regulation is associated with increased expression of Fra-1 and JunD, activator protein-1 transcription factors known to be required for involucrin expression. Altogether, these results provide insights into Pyk2 signaling in epidermis and reveal a novel role for Pyk2 in regulation of keratinocyte differentiation.

Pyk2 Signaling Events Mediate Eosinophil Chemotaxis Along Chemoattractant Gradients In The Lung

RATIONALE: Allergen-induced eosinophil migration occurs as a consequence of the expression and establishment of chemoattractant gradients in the lung (e.g., chemokines and small molecule mediators such as leukotrienes). The migratory responses of eosinophils to these chemoattractants occur as a consequence of signaling through Gαi-coupled receptors and other downstream signaling pathways some of which include the non-receptor tyrosine kinase Pyk2. METHODS: Pyk2 expression and activity in the lungs of allergen sensitized/challenged mice (control mice were challenged with saline alone) were assessed by immunohistochemistry (IHC). In addition, Pyk2 levels were assessed ex vivo using eotaxin-1 stimulated vs. unstimulated eosinophils, including the ability to block Pyk2 mediated signaling events using cell permeable, function-blocking antibodies (MTS-12A10). RESULTS: IHC showed that the accumulating eosinophils in the lungs of allergen-treated mice express significant levels of Pyk2. Whereas Pyk2 was shown to be constitutively expressed by eosinophils, stimulation by eotaxin-1 increased Pyk2 phosphorylation. Moreover, pretreatment of purified eosinophils with the Pyk2 function-blocking antibody MTS-12A10 resulted in a significant reduction in eotaxin-1 mediated chemotaxis. CONCLUSIONS: Pyk2 is a constitutively expressed signaling effector in eosinophils that is activated as part of Gαi-coupled receptor-ligand gradient responses. Specifically, eotaxin-1 mediated eosinophil chemoattraction can be reduced by blockade of Pyk2 with cell permeable antibodies, suggesting a potential novel therapeutic option for the treatment of asthma.

Proline-rich tyrosine kinase2 is involved in F-actin organization during in vitro maturation of rat oocyte

Microfilaments (actin filaments) regulate various dynamic events during meiotic maturation. Relatively, little is known about the regulation of microfilament organization in mammalian oocytes. Proline-rich tyrosine kinase2 (Pyk2), a protein tyrosine kinase related to focal adhesion kinase (FAK) is essential in actin filaments organization. The present study was to examine the expression and localization of Pyk2, and in particular, its function during rat oocyte maturation. For the first time, by using Western blot and confocal laser scanning microscopy, we detected the expression of Pyk2 in rat oocytes and found that Pyk2 and Try402 phospho-Pyk2 were localized uniformly at the cell cortex and surrounded the germinal vesicle (GV) or the condensed chromosomes at the GV stage or after GV breakdown. At the metaphase and the beginning of anaphase, Pyk2 distributed asymmetrically both in the ooplasm and the cortex with a marked staining associated with the chromosomes and the region overlying the meiotic spindle. At telophase, Pyk2 was observed in the cleavage furrows in addition to its cortex and cytoplasm localization. The dynamics of Pyk2 were similar to that of F-actin, and this kinase was found to co-localize with microfilaments in several developmental stages during rat oocyte maturation. Microinjection of Pyk2 antibody demolished the microfilaments assembly and also inhibited the first polar body (PB1) emission. These findings suggest an important role of Pyk2 for rat oocyte maturation by regulating the organization of actin filaments.

The Effects of Anti-High Molecular Weight-Melanoma Associated Antigen (HMW-MAA) Monoclonal Antibodies (mAb) Against 11q23 Positive Acute Leukemia Cells.

Acute leukemia with 11q23 aberrations is associated with especially poor outcome to standard treatment. HMW-MAA is a membrane bound proteoglycan that has been targeted in the treatment of malignant melanoma. It is widely distributed on 11q23-positive leukemic cells and is not expressed on normal hematopoietic cells. Finally, HMW-MAA is believed to function by signaling through the focal adhesion kinase (Fak)/proline-rich tyrosine kinase 2 (Pyk2) pathways and by supporting angiogenesis via its expression on pericytes. We hypothesized that inhibition of HMW-MAA with mAb directed against specific HMW-MAA determinants would effectively limit the growth of 11q23-positive acute leukemia cells in vitro and in vivo, by affecting Fak/Pyk2 pathways. We examined an 11q23-positive acute myeloid leukemia (AML) cell line (ML-2) and leukemia cells from two patients (one AML, one acute lymphoblastic leukemia) with 11q23 aberrations. Our results demonstrate that ML-2 cells expressed only the VT68.2 and 225.28 determinants, while primary acute leukemia cells expressed VT68.2, 225.28, and 763.74 determinants. Treatment of ML-2 cells with mAb against VT68.2 and 225.28 in vitro did not affect overall proliferation but downregulated phosphorylated (p) Pyk2 expression as determined by Western blotting. Further, anti-HMW-MAA mAb treatment enhanced the anti-proliferative effect of cytarabine (y=0.7875) in vitro. Based on these results, we then evaluated the dose-response relationships and toxicities of anti-HMW-MAA mAb alone and in combination with cytarabine in subcutaneous ML-2 xenografts grown in SCID mice. A trend towards tumor inhibition was noted following high dose (1 mg/kg 2x/week) anti-225 mAb therapy but was not seen at lower (0.25 and 0.5 mg/kg) anti-225 mAb doses. Mice demonstrated no side effects or weight loss as a result of anti-225 therapy. No downregulation of pPyk2 in tumor cells was observed following anti-225 therapy at any dose. Furthermore, combination anti-225.28 HMW-MAA mAb and cytarabine treatment (at increasing cytarabine doses) did not result in improved tumor inhibition as compared to cytarabine alone (p>0.05). We then evaluated the effects of HMW-MAA anti-225.28 and anti-763.74 mAb alone or together for treatment of NOD/SCID mice engrafted with primary 11q23-positive leukemia patient cells. Combination anti-HMW-MAA mAb therapy resulted in no prolongation in survival of engrafted mice as compared to control treated animals. We were able to confirm the presence of high levels of circulating HMW-MAA mAb in the serum of animals 4 days following mAb injection. In addition, explanted ML-2 cells harvested following xenograft growth still maintained expression of the HMW-MAA determinants. We conclude that naïve anti-HMW-MAA mAb neither caused a statistically significant inhibitory effect nor enhanced the effect of cytarabine on 11q23-positive acute leukemia cells in vivo. Future studies using radiolabelled or toxin-conjugated antibodies may enhance the anti-tumor effects of targeted HMW-MAA immunotherapy for 11q23-positive acute leukemia.

The differential expression of Yersinia pseudotuberculosis adhesins determines the requirement for FAK and/or Pyk2 during bacterial phagocytosis by macrophages

Phagocytosis of Yersinia pseudotuberculosis by macrophages is initiated by interactions between host cell integrin receptors and the bacterial adhesins, invasin and YadA. Two non-receptor protein tyrosine kinases, FAK and Pyk2, have been implicated in this process. In this study, we investigated the mechanisms of activation and functional requirements for these kinases during phagocytosis. A panel of Yersinia strains that differentially express invasin and YadA were used to infect cells in which FAK and/or Pyk2 expression was reduced by RNA interference. Bacterial strains that simultaneously express invasin and YadA activated FAK and Pyk2 signalling pathways that perform non-redundant functions required for Yersinia internalization. In contrast, FAK activation was found to be sufficient for phagocytosis of bacteria expressing invasin alone, and Pyk2 activation was sufficient when YadA was expressed in the absence of invasin. Based on these data, we suggest that the activation states of FAK and Pyk2, as well as the subsequent signalling events that lead to phagocytosis, are differentially regulated through the unique mechanisms of integrin engagement utilized by invasin and YadA. These findings lend insight into the molecular events that control bacterial phagocytosis as well as other integrin-based processes such as cell adhesion and migration.

RNA interference reveals a differential role of FAK and Pyk2 in cell migration, leading edge formation and increase in focal adhesions induced by LPA in intestinal epithelial cells

In the gastrointestinal mucosa, cell migration plays a crucial role in the organization and maintenance of tissue integrity but the mechanisms involved remain incompletely understood. Here, we used small-interfering RNA (siRNA)-mediated depletion of focal adhesion kinase (FAK) protein to determine the role of FAK in wound-induced migration and cytoskeletal organization in the non-transformed intestinal epithelial cells IEC-6 and IEC-18 stimulated with the G protein-coupled receptors (GPCR) agonist lysophosphatidic acid (LPA). Treatment of these cells with FAK siRNA substantially reduced FAK expression, but did not affect the expression of proline-rich tyrosine kinase 2 (Pyk2). Knockdown of FAK protein significantly inhibited LPA-induced migration of both IEC-18 and IEC-6 cells. LPA induced reorganization of actin and microtubule cytoskeleton in the leading edge was largely inhibited in FAK siRNA-transfected IEC-18 cells. Interestingly, in contrast to the FAK-/- cells, which exhibit an increased number of prominent focal adhesions when plated on fibronectin, FAK knockdown IEC-18 cells exhibited dramatically decreased number of focal adhesions in response to both LPA and fibronectin as compared with the control cells. We also used siRNAs to knockdown Pyk2 expression without reducing FAK expression. Depletion of Pyk2 did not prevent LPA-induced migration or cytoskeletal reorganization in IEC-18 cells. In conclusion, our study shows that FAK plays a critical role in LPA-induced migration, cytoskeletal reorganization, and assembly of focal adhesions in intestinal epithelial cells whereas depletion of Pyk2 did not interfere with any of these responses elicited by LPA.

Expression and intracellular localization of Pyk2 in normal and v-src transformed chicken epiphyseal chondrocytes

The expression and localization of prolin-rich tyrosine kinase 2 (Pyk2) were studied in chick embryo epiphyseal chondrocytes. Two immunoreactive bands were detected in chondrocytes, a major band with an apparent M r of 123 kDa and a minor band with an apparent M r of 68 kDa. The major band appears to migrate as a doublet with apparent M r of 116/123 kDa. Increased levels of the three forms of Pyk2 were observed in v-src transformed chondrocytes as compared to control uninfected chondrocytes. Immunofluorescent staining shows that Pyk2 is clearly visible in the cytosol and in the perinuclear region of control and v-src-chondrocytes and displays a pattern very similar to the distribution of the mitochondrial marker Mito Tracker. More, immunofluorescent staining shows that Pyk2 is nuclear in most chondrocytes. By subcellular fractionation, the p116/123 Pyk2 doublet, was found to be accumulated mainly in the cytoplasm while the p68 Pyk2 form, was found to be accumulated exclusively in the nucleus. The differential nuclear/cytoplasmic distribution of the Pyk2 forms remains unchanged after v-Src-induced transformation. The p68 Pyk2 form could no longer be detected by using a N-terminus domain-specific anti-Pyk2 antibody. Consistently, Pyk2 immunoreactivity was restricted to the cytoplasm of control and v-src transformed chondrocytes. Thus it appears that the p68 Pyk2 form that accumulates in the nucleus has a deletion in the N-terminus region.

The Tyrosine Kinase Pyk2 Promotes Migration and Invasion of Glioma Cells

Glioblastoma multiforme is extraordinarily aggressive due to the propensity of cells to migrate away from the tumor core into the surrounding normal brain. In this report, we investigated the role of proline-rich tyrosine kinase 2 (Pyk2) and FAK with regard to influencing glioma cell phenotypes. Expression of Pyk2 stimulated glioma cell migration, whereas expression of FAK inhibited glioma cell migration and stimulated cell cycle progression. Pyk2 autophosphorylation was necessary, but not sufficient, to stimulate cellular migration. The N-terminal domain of Pyk2 is required for stimulation of migration as an N-terminally deleted variant of Pyk2 failed to stimulate migration, whereas expression of an autonomous Pyk2 N-terminal domain inhibited cell migration. Substitution of the C-terminal domain of Pyk2 with the corresponding domain of FAK stimulated cell migration as effectively as wild-type Pyk2; however, substitution of the N-terminal domain of Pyk2 with that of FAK inhibited cell migration, substantiating that the N-terminal domain of Pyk2 was required to stimulate migration. Silencing of Pyk2 expression by RNA interference significantly inhibited glioma migration. Cell migration was restored on reexpression of Pyk2, but expression of FAK in Pyk2 knockdown cells failed to restore migration. We conclude that Pyk2 plays a central role in the migratory behavior of glioblastomas.

Suppression of Pyk2 attenuated the increased tyrosine phosphorylation of NMDA receptor subunit 2A after brain ischemia in rat hippocampus

Effects of suppressing the protein expression of Pyk2 on increased tyrosine phosphorylation of N-methyl- d-aspartate (NMDA) receptor subunit 2A (NR2A) after brain ischemia in rat hippocampus were studied with immunoprecipitation and immunoblot. Transient (15 min) brain ischemia and reperfusion (I/R) was induced by four-vessel occlusion in Sprague–Dawley (SD) rats. I/R led to increases of tyrosine phosphorylation of NR2A and interaction of Pyk2 and Src kinase with NR2A after 6 h of reperfusion. The increases were attenuated by Pyk2 antisense oligonucleotides intracerebroventricularly infused every 24 h for 4 days before ischemia, but not missense oligonucleotides or vehicle. The antisense also inhibited the increased auto-phosphorylation of Pyk2 and Src kinase, while the protein expression of NR2A or Src kinase had no obvious change under the above conditions. The data suggested that Pyk2 may be involved in facilitating NR2A tyrosine phosphorylation by Src kinase after I/R.

Lateral Hypothalamic Signaling Mechanisms Underlying Feeding Stimulation: Differential Contributions of Src Family Tyrosine Kinases to Feeding Triggered Either by NMDA Injection or by Food Deprivation

In rats, feeding can be triggered experimentally using many approaches. Included among these are (1) food deprivation and (2) acute microinjection of the neurotransmitter l-glutamate (Glu) or its receptor agonist NMDA into the lateral hypothalamic area (LHA). Under both paradigms, the NMDA receptor (NMDA-R) within the LHA appears critically involved in transferring signals encoded by Glu to stimulate feeding. However, the intracellular mechanisms underlying this signal transfer are unknown. Because protein-tyrosine kinases (PTKs) participate in NMDA-R signaling mechanisms, we determined PTK involvement in LHA mechanisms underlying both types of feeding stimulation through food intake and biochemical measurements. LHA injections of PTK inhibitors significantly suppressed feeding elicited by LHA NMDA injection (up to 69%) but only mildly suppressed deprivation feeding (24%), suggesting that PTKs may be less critical for signals underlying this feeding behavior. Conversely, food deprivation but not NMDA injection produced marked increases in apparent activity for Src PTKs and in the expression of Pyk2, an Src-activating PTK. When considered together, the behavioral and biochemical results demonstrate that, although it is easier to suppress NMDA-elicited feeding by PTK inhibitors, food deprivation readily drives PTK activity in vivo. The latter result may reflect greater PTK recruitment by neurotransmitter receptors, distinct from the NMDA-R, that are activated during deprivation-elicited but not NMDA-elicited feeding. These results also demonstrate how the use of only one feeding stimulation paradigm may fail to reveal the true contributions of signaling molecules to pathways underlying feeding behavior in vivo.

Expression of tyrosine kinases FAK and Pyk2 in 331 human astrocytomas.

The progression of malignancy from astrocytomas to glioblastomas remains clinically as well as histopathologically unpredictable. The focal adhesion kinase (FAK) and the proline-rich tyrosine kinase (Pyk2) show a high expression in glioma cell lines and have an influence on increased cell proliferation and migration of glioma cells in vitro and in vivo. The aim of this study was to correlate the coexpression of FAK and Pyk2 to the WHO grade of malignancy in human astrocytomas. Immunohistochemical staining scores of FAK and Pyk2 were analyzed in 331 astrocytomas and correlated to each other and to the WHO grade. Significant coexpression of FAK and Pyk2 in astrocytomas was demonstrated. Pyk2 expression occurred much more frequently and with higher expression scores within the different WHO grades. Beyond this, a significant correlation between the WHO grade of malignancy of astrocytomas and the expression of FAK, as well as of Pyk2, was detected. This connection and the roles of these two tyrosine kinases in the progression of tumors should be confirmed by further studies.

Differential regulation of cell migration and proliferation through proline-rich tyrosine kinase 2 in endothelial cells.

Proline-rich tyrosine kinase 2 (Pyk2), a member of the focal adhesion kinase family, is thought to act as a key component in vasculogenesis and angiogenesis. Therefore, we studied the effect of mutant Pyk2 expression on the migration and proliferation in endothelial cells (ECs). Two types of mutant Pyk2 were examined by adenovirus vectors AxCA-Pyk2K457A, expressing a kinase inactive mutant, and AxCA-Pyk2Y402F, expressing a tyrosine autophosphorylation site mutant, in addition to AxCA-Pyk2, expressing wild-type Pyk2. Migration of ECs infected with AxCA-Pyk2Y402F increased to a level similar to that of ECs infected with AxCA-Pyk2. The size of effect was dependent on the amount of applied adenoviruses within the range of 3-30 multiplicity of infection. In contrast, AxCA-Pyk2K457A infection did not show any significant effect on cell migration. Western blotting showed that both phosphorylation of Pyk2 Y(881) and association of p130(Cas) with Pyk2 were enhanced in ECs infected with AxCA-Pyk2Y402F as well as with AxCA-Pyk2, but not in ECs infected with AxCA-Pyk2K457A. Therefore, signaling mediated by Pyk2 Y(881) and p130(Cas) may be involved in the migration of ECs infected either with AxCA-Pyk2Y402F or with AxCA-Pyk2. In proliferation assay, AxCA-Pyk2 infection suppressed EC proliferation significantly; however, neither AxCA-Pyk2Y402F nor AxCA-Pyk2K457A showed such an inhibitory effect. Thus, the two Pyk2 mutants revealed that Pyk2 signaling differentially regulates cell migration and proliferation pathways.

Pyk2 Amplifies Epidermal Growth Factor and c-Src-induced Stat3 Activation

Signal transducers and activators of transcription factors (STATs) mediate many of the cellular responses that occur following cytokine, growth factor, and hormone signaling. STATs are activated by tyrosine and serine phosphorylation, which normally occurs as a tightly regulated process. Dysregulated STAT activity may facilitate oncogenesis, as constitutively activated STATs have been found in many human tumors as well as in v-abl- and v-src-transformed cell lines. Pyk2 is a member of the focal adhesion kinase family and can be activated by c-Src, epidermal growth factor receptor (EGFR), Janus kinase 1, tyrosine kinases, and G-protein-coupled receptor signaling. Although Pyk2 has been implicated in Janus kinase-dependent activation of MAPK and Stat1, no role for Pyk2 in the activation of other STAT proteins has been ascribed. Here, we provide evidence that Pyk2, along with c-Src, facilitates EGFR-mediated Stat3 activation. Pyk2 expression in HeLa cells induces Stat3 reporter gene activation and Stat3 phosphorylation on amino acid residues Tyr-705 and Ser-727. Together Pyk2 and c-Src potently activate Stat3, and Pyk2 enhances Stat3-induced cell proliferation. Moreover, the expression of a dominant negative version of Pyk2 impairs c-Src-induced Stat3 activation and cell proliferation. The treatment of A431 cells with EGF results in the recruitment of c-Src, Pyk2, and Stat3 to the EGFR and the phosphorylation of c-Src, Pyk2, and Stat3. Expression of constructs for dominant negative forms of either Pyk2 or c-Src impair EGF-induced Stat3 phosphorylation. These results indicate that Pyk2 facilitates EGFR- and c-Src-mediated Stat3 activation, thereby implicating Pyk2 activation as a potential co-mediator in triggering Stat3-induced oncogenesis.

P210BCR/ABL-induced alteration of pre-mRNA splicing in primary human CD34+ hematopoietic progenitor cells.

Chronic myelogenous leukemia (CML) is a malignancy of the human hematopoietic stem cell (HSC) caused by the p210BCR/ABL oncoprotein. Although alternative splicing of pre-mRNA is a critical determinant of a cell's protein repertoire, it has not been associated with CML pathogenesis. We identified a BCR/ABL-dependent increase in expression of multiple genes involved in pre-mRNA splicing (eg SRPK1, RNA Helicase II/Gu, and hnRNPA2/B1) by subtractive hybridization of cDNA from p210BCR/ABL-eGFP vs eGFP-transduced umbilical cord blood CD34+ cells. beta1-integrin signaling is important to HSC maintenance and proliferation/differentiation, and is abnormal in CML. As an example of how changes in pre-mRNA processing might contribute to CML pathogenesis, we observed alternative splicing of a gene for a beta1-integrin-responsive nonreceptor tyrosine kinase (PYK2), resulting in increased expression of full-length Pyk2 in BCR/ABL-containing cells. Treatment of p210BCR/ABL-positive cells with the Abl-specific tyrosine kinase inhibitor STI571 reverted PYK2 splicing to a configuration more consistent with normal cells, and correlated with decreased expression of BCR/ABL-induced proteins involved in pre-mRNA processing. Whether altered PYK2 splicing contributes to CML pathogenesis remains undetermined; however, we propose that generic changes in pre-mRNA splicing as a result of p210BCR/ABL kinase activity may contribute to CML pathogenesis.

Antidepressant effect of the calcium-activated tyrosine kinase Pyk2 in the lateral septum

Background Accumulating evidence indicates that neural activity in the lateral septum (LS) influences the pathophysiology of depression and therapeutic effectiveness of antidepressant drugs. For example, the development of behavioral deficits in animal screens for antidepressant drug activity corresponds with a blunting of LS activity, whereas chronic treatment with antidepressants enhances cell firing in the LS; however, the molecular mechanisms underlying such behavioral functions of the LS have not been determined. The nonreceptor tyrosine kinase Pyk2 is highly expressed in the LS and plays important roles in regulating cellular excitability and synaptic plasticity, making it an attractive candidate for regulating the effects of stress and antidepressants on LS functioning and behavior. We provide evidence that stress decreases Pyk2 phosphorylation in the LS, whereas enhancing Pyk2 expression in LS neurons has an antidepressant effect behaviorally. Methods Pyk2 messenger ribonucleic acid (mRNA) expression in the rat forebrain was detected by in situ hybridization, and a brief description of the distribution of Pyk2 mRNA in selected areas is presented. Levels of total Pyk2 protein and phosphorylated Pyk2 were subsequently measured in the LS and hippocampus following stress exposure, as were levels of extracellular stimuli-regulated kinase (Erk) and phospho-Erk. Herpes simplex virus (HSV)-mediated gene transfer was then used to enhance Pyk2 expression in the LS, and the effect this had on behavior in the learned helplessness model of depression was evaluated. Results High levels of Pyk2 mRNA were detected in a number of forebrain regions, including the hippocampus and LS. Following acute stress exposure, subjects showed a decrease in phosphorylated Pyk2 and Erk in the LS but not in the hippocampus. Total levels of Pyk2 and Erk remained unchanged following stress. In the learned helplessness paradigm, injection of HSV-Pyk2 into the LS prevented the active avoidance deficit caused by exposure to inescapable shock, indicative of an antidepressant effect. Conclusions These results indicate that following acute stress, Pyk2 and Erk activity in the LS are decreased, whereas experimentally increasing Pyk2 activity in LS neurons reverses the behavioral deficits of acute, inescapable stress. These findings establish a role for the tyrosine kinase Pyk2 in the biochemical and behavioral responses to stress and suggest a possible role in the pathophysiology of depression, particularly notable considering Pyk2’s role in promoting synaptic plasticity.

Proline-rich tyrosine kinase 2 regulates proliferation and differentiation of prostate cells

Proline-rich tyrosine kinase 2 (Pyk2) expression in prostate epithelium inversely correlated with degree of malignancy of prostate cancers, thus the role of Pyk2 in the regulation of prostate cells proliferation and differentiation was investigate in PC3 cells. Pyk2 can be activated by canonic stimuli such as tumor necrosis factorα and lysophosphatidic acid (LPA) in PC3 cells, in addition, LPA stimulated Pyk2 phosphorylation also induced extracellular signal-regulated kinase 1 and 2 activation in these cells. Proliferation of PC3 cell clones (PC3-PKM) expressing a dominant negative kinase-defective Pyk2 mutant is consistently decreased in respect to that of wild type PC3 cells. In addition, PC3-PKM clones underwent total block cell proliferation upon treatment with dibutyryl cAMP. Finally, in the presence of sustained levels of intracellular cAMP, PC3-PKM cells, but not wild type PC3 cells, acquired a neuron-like morphology. Taken together our results suggest that Pyk2 plays a role in the regulation of prostate cell proliferation and, more interestingly, its expression may represents a sensitive marker of prostate state of differentiation.

PYK2 Expression and Phosphorylation in Neonatal and Adult Cardiomyocytes

A. L. Bayer, A. G. Ferguson, P. A. Lucchesi and A. M. Samarel. PYK2 Expression and Phosphorylation in Neonatal and Adult Cardiomyocytes. Journal of Molecular and Cellular Cardiology (2001) 33, 1017–1030. Proline-rich tyrosine kinase (PYK2) is a Ca2+-dependent, non-receptor protein tyrosine kinase involved in growth factor signaling. Although PYK2 is expressed in a variety of tissues, it has not yet been identified in cardiac muscle. Therefore, immunocytochemical and Western blotting techniques were used to examine PYK2 expression and phosphorylation in neonatal and adult rat ventricular cardiomyocytes (NRVM and ARVM, respectively). PYK2 concentration was much greater in neonatal, than in adult ventricular tissue and cardiomyocytes. In cultured cells, PYK2 expression was highly dependent on [Ca2+]itransients and contractile activity. Non-contracting, low-density NRVM in serum-free culture expressed very low levels of PYK2, while high-density, spontaneously contracting NRVM showed a ∼12-fold increase in PYK2 expression. Conversely, high-density NRVM treated with nifedipine (10 μM, 48 h) to block spontaneous [Ca2+]itransients and contractile activity resulted in a 2.6-fold decrease in PYK2 levels. Similarly, overnight culture of quiescent ARVM markedly reduced PYK2 levels. Chronic treatment (48 h) of cultured NRVM with the hypertrophic agonist endothelin-1 (ET) (10–300 n M) did not significantly increase PYK2 levels, but strongly shifted the ratio of phosphorylated to total PYK2, indicating that PYK2 phosphorylation accompanies cardiomyocyte hypertrophy. Endothelin-1 also acutely activated PYK2 in both cultured NRVM, and in freshly isolated ARVM. These results suggest that PYK2 is involved in the generation of certain aspects of cardiomyocyte hypertrophy.

Regulation of Pyk2 expression by p56 Lck in Jurkat T lymphocytes

Lysates from the Jurkat T lymphocyte cell line were immunoblotted with anti-Pyk2, and two major forms of Pyk2 were identified. When lysates from the p56 Lck negative (J.CaM1/Rep3) and CD45 negative Jurkat cell line derivatives were immunoblotted with anti-Pyk2, only the lower mobility form of Pyk2 was predominant. Transfection of J.CaM1 cells with p56 Lck restored expression of the multiple forms of Pyk2. Using RT-PCR, we found that both species of the alternatively spliced mRNA for Pyk2 were present in all of the lines regardless of their ability to express CD45 or p56 Lck protein. When p56 Lck immunoprecipitates were immunoblotted with anti-Pyk2, only the higher mobility form of Pyk2 immunoprecipitated with p56 Lck. These data demonstrate that certain members of the Src family of kinases interact preferentially with the different isoforms of Pyk2 and may have a role in the regulation of the Pyk2 protein in lymphocytes.

Variations of Proline-Rich Kinase Pyk2 Expression Correlate with Prostate Cancer Progression

Proline-rich kinase 2 (Pyk2), also known as CAKβ (cell adhesion kinase β), is a cytoplasmic tyrosine kinase that is structurally related to focal adhesion kinase. Pyk2 is expressed in different cell types including brain cells, fibroblasts, platelets, and other hemopoietic cells. Pyk2 is rapidly tyrosine phosphorylated in response to diverse extracellular signals acting via different post receptor pathways. We have investigated whether this protein kinase is functionally expressed in normal and neoplastic prostate tissues. In this study, we demonstrate that Pyk2 is expressed only in normal epithelial prostate tissue and in benign prostatic hyperplasia, whereas its expression progressively declines with an increasing grade of malignancy of prostate cancer.