Podosome Nucleation Is Facilitated by Multivalent Interactions between Syk and ITAM-containing Membrane Complexes.

Immune cells survey their microenvironment by forming dynamic cellular protrusions that enable chemotaxis, contacts with other cells, and phagocytosis. Podosomes are a unique type of protrusion structured by an adhesive ring of active integrins that surround an F-actin-rich core harboring degradative proteases. Although the features of podosomes, once-established, have been well defined, the steps that lead to podosome formation remain poorly understood by comparison. In this study, we report that spleen tyrosine kinase (Syk) is a critical regulator of podosome formation. Deletion of Syk or targeting its kinase activity eliminated the ability for murine macrophages to form podosomes. We found that the kinase activity of Syk was important for the phosphorylation of its substrates, HS1 and Pyk2, both of which regulate podosome formation. Additionally, before podosomes form, we report that the tandem Src homology 2 domains of Syk afforded multivalent clustering of ITAM-containing adaptors that associated with integrins to structure platforms that initiate podosomes. We therefore propose that Syk has a dual role in regulating podosomes: first, by facilitating the assembly of multivalent signaling hubs that nucleate their formation and second, by sustaining tyrosine kinase activity of the podosomes once they form against their substrates. In cells expressing recently identified gain-of-function variants of SYK, podosomes were dysregulated. These results implicate SYK in the (patho)physiological functions of podosomes in macrophages.

Integrin αVß1-activated PYK2 promotes the progression of non-small-cell lung cancer via the STAT3-VGF axis.

BACKGROUND: Non-small-cell lung cancer (NSCLC) accounts for 80-85% of all lung cancer and is the leading cause of cancer-related deaths globally. Although various treatment strategies have been introduced, the 5-year survival rate of patients with NSCLC is only 20-30%. Thus, it remains necessary to study the pathogenesis of NSCLC and develop new therapeutic drugs. Notably, PYK2 has been implicated in the progression of many tumors, including NSCLC, but its detailed mechanism remains unclear. In this study, we aimed to elucidate the mechanisms through which PYK2 promotes NSCLC progression. METHODS: The mRNA and protein levels of various molecules were measured using qRT-PCR, western blot (WB), and immunohistochemistry (IHC), respectively. We established stable PYK2 knockdown and overexpression cell lines, and CCK-8, EdU, and clonogenic assays; wound healing, transwell migration, and Matrigel invasion assays; and flow cytometry were employed to assess the phenotypes of tumor cells. Protein interactions were evaluated with co-immunoprecipitation (co-IP), immunofluorescence (IF)-based colocalization, and nucleocytoplasmic separation assays. RNA sequencing was performed to explore the transcriptional regulation mediated by PYK2. Secreted VGF levels were examined using ELISA. Dual-luciferase reporter system was used to detect transcriptional regulation site. PF4618433 (PYK2 inhibitor) and Stattic (STAT3 inhibitor) were used for rescue experiments. A public database was mined to analyze the effect of these molecules on NSCLC prognosis. To investigate the role of PYK2 in vivo, mouse xenograft models of lung carcinoma were established and examined. RESULTS: The protein level of PYK2 was higher in human NSCLC tumors than in the adjacent normal tissue, and higher PYK2 expression was associated with poorer prognosis. PYK2 knockdown inhibited the proliferation and motility of tumor cells and caused G1-S arrest and cyclinD1 downregulation in A549 and H460 cells. Meanwhile, PYK2 overexpression had the opposite effect in H1299 cells. The siRNA-induced inhibition of integrins alpha V and beta 1 led to the downregulation of p-PYK2(Tyr402). Activated PYK2 could bind to STAT3 and enhance its phosphorylation at Tyr705, regulating the nuclear accumulation of p-STAT3(Tyr705). This further promoted the expression of VGF, as confirmed by RNA sequencing in a PYK2-overexpressing H1299 cell line and validated by rescue experiments. Two sites in promoter region of VGF gene were confirmed as binding sites of STAT3 by Dual-luciferase assay. Data from the TGCA database showed that VGF was related to the poor prognosis of NSCLC. IHC revealed higher p-PYK2(Tyr402) and VGF expression in lung tumors than in adjacent normal tissues. Moreover, both proteins showed higher levels in advanced TNM stages than earlier ones. A positive linear correlation existed between the IHC score of p-PYK2(Tyr402) and VGF. Knockdown of VGF inhibited tumor progression and reversed the tumor promoting effect of PYK2 overexpression in NSCLC cells. Finally, the mouse model exhibited enhanced tumor growth when PYK2 was overexpressed, while the inhibitors PF4618433 and Stattic could attenuate this effect. CONCLUSIONS: The Integrin αVß1-PYK2-STAT3-VGF axis promotes NSCLC development, and the PYK2 inhibitor PF4618433 and STAT3 inhibitor Stattic can reverse the pro-tumorigenic effect of high PYK2 expression in mouse models. Our findings provide insights into NSCLC progression and could guide potential therapeutic strategies against NSCLC with high PYK2 expression levels.

Mitochondrial translation failure represses cholesterol gene expression via Pyk2-Gsk3ß-Srebp2 axis.

Neurodegenerative diseases and other age-related disorders are closely associated with mitochondrial dysfunction. We previously showed that mice with neuron-specific deficiency of mitochondrial translation exhibit leukoencephalopathy because of demyelination. Reduced cholesterol metabolism has been associated with demyelinating diseases of the brain such as Alzheimer's disease. However, the molecular mechanisms involved and relevance to the pathogenesis remained unknown. In this study, we show that inhibition of mitochondrial translation significantly reduced expression of the cholesterol synthase genes and degraded their sterol-regulated transcription factor, sterol regulatory element-binding protein 2 (Srebp2). Furthermore, the phosphorylation of Pyk2 and Gsk3ß was increased in the white matter of p32cKO mice. We observed that Pyk2 inhibitors reduced the phosphorylation of Gsk3ß and that GSK3ß inhibitors suppressed degradation of the transcription factor Srebp2. The Pyk2-Gsk3ß axis is involved in the ubiquitination of Srebp2 and reduced expression of cholesterol gene. These results suggest that inhibition of mitochondrial translation may be a causative mechanism of neurodegenerative diseases of aging. Improving the mitochondrial translation or effectiveness of Gsk3ß inhibitors is a potential therapeutic strategy for leukoencephalopathy.

PYK2, a hub of signaling networks in breast cancer progression.

Breast cancer (BC) involves complex signaling networks characterized by extensive cross-communication and feedback loops between and within multiple signaling cascades. Many of these signaling pathways are driven by genetic alterations of oncogene and/or tumor-suppressor genes and are influenced by various environmental cues. We describe unique roles of the non-receptor tyrosine kinase (NRTK) PYK2 in signaling integration and feedback looping in BC. PYK2 functions as a signaling hub in various cascades, and its involvement in positive and negative feedback loops enhances signaling robustness, modulates signaling dynamics, and contributes to BC growth, epithelial-to-mesenchymal transition (EMT), stemness, migration, invasion, and metastasis. We also discuss the potential of PYK2 as a therapeutic target in various BC subtypes.

Thrombin-Induced COX-2 Expression and PGE2 Synthesis in Human Tracheal Smooth Muscle Cells: Role of PKCδ/Pyk2-Dependent AP-1 Pathway Modulation.

In this study, we confirmed that thrombin significantly increases the production of COX-2 and PGE2 in human tracheal smooth muscle cells (HTSMCs), leading to inflammation in the airways and lungs. These molecules are well-known contributors to various inflammatory diseases. Here, we investigated in detail the involved signaling pathways using specific inhibitors and small interfering RNAs (siRNAs). Our results demonstrated that inhibitors targeting proteins such as protein kinase C (PKC)δ, proline-rich tyrosine kinase 2 (Pyk2), c-Src, epidermal growth factor receptor (EGFR), phosphatidylinositol 3-kinase (PI3K), or activator protein-1 (AP-1) effectively reduced thrombin-induced COX-2 and PGE2 production. Additionally, transfection with siRNAs against PKCδ, Pyk2, c-Src, EGFR, protein kinase B (Akt), or c-Jun mitigated these responses. Furthermore, our observations revealed that thrombin stimulated the phosphorylation of key components of the signaling cascade, including PKCδ, Pyk2, c-Src, EGFR, Akt, and c-Jun. Thrombin activated COX-2 promoter activity through AP-1 activation, a process that was disrupted by a point-mutated AP-1 site within the COX-2 promoter. Finally, resveratrol (one of the most researched natural polyphenols) was found to effectively inhibit thrombin-induced COX-2 expression and PGE2 release in HTSMCs through blocking the activation of Pyk2, c-Src, EGFR, Akt, and c-Jun. In summary, our findings demonstrate that thrombin-induced COX-2 and PGE2 generation involves a PKCδ/Pyk2/c-Src/EGFR/PI3K/Akt-dependent AP-1 activation pathway. This study also suggests the potential use of resveratrol as an intervention for managing airway inflammation.

Pyk2/FAK Signaling Is Upregulated in Recurrent Glioblastoma Tumors in a C57BL/6/GL261 Glioma Implantation Model.

The majority of glioblastomas (GBMs) recur shortly after tumor resection and recurrent tumors differ significantly from newly diagnosed GBMs, phenotypically and genetically. In this study, using a Gl261-C57Bl/6 mouse glioma implantation model, we identified significant upregulation of proline-rich tyrosine kinase Pyk2 and focal adhesion kinase (FAK) phosphorylation levels-pPyk2 (579/580) and pFAK (925)-without significant modifications in total Pyk2 and FAK protein expression in tumors regrown after surgical resection, compared with primary implanted tumors. Previously, we demonstrated that Pyk2 and FAK are involved in the regulation of tumor cell invasion and proliferation and are associated with reduced overall survival. We hypothesized that the use of inhibitors of Pyk2/FAK in the postsurgical period may reduce the growth of recurrent tumors. Using Western blot analysis and confocal immunofluorescence approaches, we demonstrated upregulation of Cyclin D1 and the Ki67 proliferation index in tumors regrown after resection, compared with primary implanted tumors. Treatment with Pyk2/FAK inhibitor PF-562271, administered through oral gavage at 50 mg/kg daily for two weeks beginning 2 days before tumor resection, reversed Pyk2/FAK signaling upregulation in recurrent tumors, reduced tumor volume, and increased animal survival. In conclusion, the use of Pyk2/FAK inhibitors can contribute to a delay in GBM tumor regrowth after surgical resection.

PF-431396 hydrate inhibition of kinase phosphorylation during adherent-invasive Escherichia coli infection inhibits intra-macrophage replication and inflammatory cytokine release.

Adherent-invasive Escherichia coli (AIEC) have been implicated in the aetiology of Crohn's disease (CD). They are characterized by an ability to adhere to and invade intestinal epithelial cells, and to replicate intracellularly in macrophages resulting in inflammation. Proline-rich tyrosine kinase 2 (PYK2) has previously been identified as a risk locus for inflammatory bowel disease and a regulator of intestinal inflammation. It is overexpressed in patients with colorectal cancer, a major long-term complication of CD. Here we show that Pyk2 levels are significantly increased during AIEC infection of murine macrophages while the inhibitor PF-431396 hydrate, which blocks Pyk2 activation, significantly decreased intramacrophage AIEC numbers. Imaging flow cytometry indicated that Pyk2 inhibition blocked intramacrophage replication of AIEC with no change in the overall number of infected cells, but a significant reduction in bacterial burden per cell. This reduction in intracellular bacteria resulted in a 20-fold decrease in tumour necrosis factor α secretion by cells post-AIEC infection. These data demonstrate a key role for Pyk2 in modulating AIEC intracellular replication and associated inflammation and may provide a new avenue for future therapeutic intervention in CD.

PTK2B regulates immune responses of neutrophils and protects mucosal inflammation in ulcerative colitis.

Neutrophils participate in the pathogenesis of ulcerative colitis (UC) through regulating the intestinal homeostasis. Several inflammatory diseases are reported to be regulated by proline-rich tyrosine kinase 2B (PTK2B). However, the role of PTK2B in regulating the function of neutrophils and the pathogenesis of UC remains unknown. In this study, the mRNA and protein levels of PTK2B in the colonic tissues from UC patients were measured by using quantitative real-time polymerase chain reaction (qRT-PCR), western blotting, and immunohistochemistry. TAE226, a PTK2B inhibitor, was used to inhibit the activity of PTK2B in neutrophils, and then, the pro-inflammatory factors were analyzed by using qRT-PCR and ELISA. To determine the role of PTK2B in intestinal inflammation, a dextran sulfate sodium (DSS)-induced colitis model was established in PTK2B gene knockout (PTK2B KO) and wild-type (WT) mice. We found that compared with healthy donor controls, the expression level of PTK2B was significantly elevated in inflamed mucosa from UC patients. In addition, expression of PTK2B was positively correlated with the severity of disease. Pharmacological inhibition of PTK2B could markedly reduce the generation of reactive oxygen species (ROS), myeloperoxidase (MPO), and antimicrobial peptides (S100a8 and S100a9) in neutrophils. The vitro study showed that tumor necrosis factor (TNF)-α is involved in promoting the expression of PTK2B in neutrophils. As expected, UC patients treated with infliximab, an anti-TNF-α agent, showed significantly reduced level of PTK2B in neutrophils, as well as in the intestinal mucosa. Of note, compared with DSS-treated WT mice, DSS-treated PTK2B KO mice showed more severe colitis symptoms. Mechanistically, PTK2B could enhance neutrophil migration by regulating CXCR2 and GRK2 expression via the p38 MAPK pathway. Additionally, mice treated with TAE226 exhibited the same effects. In conclusion, PTK2B is involved in the pathogenesis of UC by promoting the migration of neutrophils and inhibiting mucosal inflammation, highlighting PTK2B as a new potential therapeutic target to treat UC.

The role and molecular mechanism of metabolic reprogramming of colorectal cancer by UBR5 through PYK2 regulation of OXPHOS expression study.

Colorectal carcinoma (CRC) is the third most malignant tumor in the world, but the key mechanisms of CRC progression have not been confirmed. UBR5 and PYK2 expression levels were detected by RT-qPCR. The levels of UBR5, PYK2, and mitochondrial oxidative phosphorylation (OXPHOS) complexes were detected by western blot analysis. Flow cytometry was used to detect ROS activity. The CCK-8 assay was used to assess cell proliferation and viability. The interaction between UBR5 and PYK2 was detected by immunoprecipitation. A clone formation assay was used to determine the cell clone formation rate. The ATP level and lactate production of each group of cells were detected by the kit. EdU staining was performed for cell proliferation.Transwell assay was performed for cell migration ability. For the CRC nude mouse model, we also observed and recorded the volume and mass of tumor-forming tumors. The expression of UBR5 and PYK2 was elevated in both CRC and human colonic mucosal epithelial cell lines, and knockdown of UBR5 had inhibitory effects on cancer cell proliferation and cloning and other behaviors in the CRC process by knockdown of UBR5 to downregulate the expression of PYK2, thus inhibiting the OXPHOS process in CRC; rotenone (OXPHOS inhibitor) treatment enhanced all these inhibitory effects. Knockdown of UBR5 can reduce the expression level of PYK2, thus downregulating the OXPHOS process in CRC cell lines and inhibiting the CRC metabolic reprogramming process.

Activation loop phosphorylation tunes conformational dynamics underlying Pyk2 tyrosine kinase activation.

Pyk2 is a multidomain non-receptor tyrosine kinase that undergoes a multistage activation mechanism. Activation is instigated by conformational rearrangements relieving autoinhibitory FERM domain interactions. The kinase autophosphorylates a central linker residue to recruit Src kinase. Pyk2 and Src mutually phosphorylate activation loops to confer full activation. While the mechanisms of autoinhibition are established, the conformational dynamics associated with autophosphorylation and Src recruitment remain unclear. We employ hydrogen/deuterium exchange mass spectrometry and kinase activity profiling to map the conformational dynamics associated with substrate binding and Src-mediated activation loop phosphorylation. Nucleotide engagement stabilizes the autoinhibitory interface, while phosphorylation deprotects both FERM and kinase regulatory surfaces. Phosphorylation organizes active site motifs linking catalytic loop with activation segment. Dynamics of the activation segment anchor propagate to EF/G helices to prevent reversion of the autoinhibitory FERM interaction. We employ targeted mutagenesis to dissect how phosphorylation-induced conformational rearrangements elevate kinase activity above the basal autophosphorylation rate.

PYK2 is overexpressed in chronic lymphocytic leukaemia: A potential new therapeutic target.

Chronic Lymphocytic Leukaemia (CLL) is the most common adult B-cell leukaemia and despite improvement in patients' outcome, following the use of targeted therapies, it remains incurable. CLL supportive microenvironment plays a key role in both CLL progression and drug resistance through signals that can be sensed by the main components of the focal adhesion complex, such as FAK and PYK2 kinases. Dysregulations of both kinases have been observed in several metastatic cancers, but their role in haematological malignancies is still poorly defined. We characterized FAK and PYK2 expression and observed that PYK2 expression is higher in leukaemic B cells and its overexpression significantly correlates with their malignant transformation. When targeting both FAK and PYK2 with the specific inhibitor defactinib, we observed a dose-response effect on CLL cells viability and survival. In vivo treatment of a CLL mouse model showed a decrease of the leukaemic clone in all the lymphoid organs along with a significant reduction of macrophages and of the spleen weight and size. Our results first define a possible prognostic value for PYK2 in CLL, and show that both FAK and PYK2 might become putative targets for both CLL and its microenvironment (e.g. macrophages), thus paving the way to an innovative therapeutic strategy.

Long Non-Coding RNA LINC00052 Targets miR-548p/Notch2/Pyk2 to Modulate Tumor Budding and Metastasis of Human Breast Cancer.

Abnormal expression of long non-coding RNAs (lncRNAs) is involved in many pathological processes of cancers. However, the role of lncRNA LINC00052 in breast cancer progression is still unclear. Here, LINC00052 expression was detected by in situ hybridization and quantitative real-time PCR assays. Cell Counting Kit-8, wound healing, and transwell assays were used to investigate changes in the proliferation, migration, and invasion of breast cancer cells. MiR-548p was found associated with LINC00052 or Notch2 by RNA pull-down, dual-luciferase reporter, and qRT-PCR assays. The effect of LINC00052 on lung metastasis was explored through in vivo experiments. High LINC00052 expression was observed in breast cancer tissues and cells. LINC00052 silencing inhibited the proliferation, migration, and invasion of MCF7 cells, and LINC00052 overexpression produced the opposite results. MiR-548p, a target gene of LINC00052, partially rescued the effects of LINC00052 on proliferation, migration, and invasion of MCF7. Notch2 was the target of miR-548p and LINC00052 could promote Notch2 expression. Moreover, the phosphorylation of proline-rich tyrosine kinase 2 (Pyk2), a downstream factor of Notch2, was increased by LINC00052, and a Pyk2 mutant could inhibit the cell migration and invasion induced by LINC00052 overexpression in MDA-MB-468 cells, which was similar to the function of the miR-548p mimic. We further demonstrated that LINC00052 exacerbated the metastases of breast cancer cells in vivo. Our research demonstrated that LINC00052 is highly expressed in breast cancer and promotes breast cancer proliferation, migration, and invasion via the miR-548p/Notch2/Pyk2 axis. LINC00052 could serve as a potential therapeutic target for breast cancer.

Microglia PTK2B/Pyk2 in the Pathogenesis of Alzheimer's Disease.

Alzheimer's disease (AD) is a highly hereditary disease with complex genetic susceptibility factors. Extensive genome-wide association studies have established a distinct susceptibility link between the protein tyrosine kinase 2ß (PTK2B) gene and late-onset Alzheimer's disease (LOAD), but the specific pathogenic mechanisms remain incompletely understood. PTK2B is known to be expressed in neurons, and recent research has revealed its more important significance in microglia. Elucidating the role of PTK2B high expression in microglia in AD's progression is crucial for uncovering novel pathogenic mechanisms of the disease. Our review of existing studies suggests a close relationship between PTK2B/proline-rich tyrosine kinase 2 (Pyk2) and tau pathology, and this process might be ß-amyloid (Aß) dependence. Pyk2 is hypothesized as a pivotal target linking Aß and tau pathologies. Concurrently, Aß-activated Pyk2 participates in the regulation of microglial activation and its proinflammatory functions. Consequently, it is reasonable to presume that Pyk2 in microglia contributes to amyloid-induced tau pathology in AD via a neuroinflammatory pathway. Furthermore, many things remain unclear, such as identifying the specific pathways that lead to the release of downstream inflammatory factors due to Pyk2 phosphorylation and whether all types of inflammatory factors can activate neuronal kinase pathways. Additionally, further in vivo experiments are essential to validate this hypothesized pathway. Considering PTK2B/Pyk2's potential role in AD pathogenesis, targeting this pathway may offer innovative and promising therapeutic approaches for AD.

High expression of PYK2 is associated with poor prognosis and cancer progression in early-stage cervical carcinoma.

Proline-rich tyrosine kinase-2 (PYK2), also known as calcium dependent tyrosine kinase, regulates different signal transduction cascades that control cell proliferation, migration, and invasion. However, the role of PYK2 in cervical cancer remains to be elucidated. The current study retrospectively included 134 patients with cervical cancer from December 2007 to September 2014. PYK2 expression was detected in tissue microarray and cervical cancer cell lines. Statistical analysis was performed to evaluate its clinicopathological significance. Small interfering RNA (siRNA) was employed to suppress endogenous PYK2 expression in cervical cancer cells to observe the biological function. PYK2 expression was up-regulated in cervical cancer specimens compared with paired adjacent normal cervical tissue samples. Statistical analyses indicated that PYK2 expression might be an independent prognostic indicator for patients with early-stage cervical cancer. A nomogram model was constructed based on PYK2 expression and other clinicopathological risk factors, and it performed well in predicting patients survival. In cellular studies, down-regulation of PYK2 remarkably inhibited cellular proliferation, migration and invasion. PYK2 expression possessed the potential to serve as a novel prognostic marker in cervical cancer patients.

Protein tyrosine kinase 2b inhibition reverts niche-associated resistance to tyrosine kinase inhibitors in AML.

FLT3 tyrosine kinase inhibitor (TKI) therapy evolved into a standard therapy in FLT3-mutated AML. TKI resistance, however, develops frequently with poor outcomes. We analyzed acquired TKI resistance in AML cell lines by multilayered proteome analyses. Leupaxin (LPXN), a regulator of cell migration and adhesion, was induced during early resistance development, alongside the tyrosine kinase PTK2B which phosphorylated LPXN. Resistant cells differed in cell adhesion and migration, indicating altered niche interactions. PTK2B and LPXN were highly expressed in leukemic stem cells in FLT3-ITD patients. PTK2B/FAK inhibition abrogated resistance-associated phenotypes, such as enhanced cell migration. Altered pathways in resistant cells, assessed by nascent proteomics, were largely reverted upon PTK2B/FAK inhibition. PTK2B/FAK inhibitors PF-431396 and defactinib synergized with different TKIs or daunorubicin in FLT3-mutated AML. Midostaurin-resistant and AML cells co-cultured with mesenchymal stroma cells responded particularly well to PTK2B/FAK inhibitor addition. Xenograft mouse models showed significant longer time to leukemia symptom-related endpoint upon gilteritinib/defactinib combination treatment in comparison to treatment with either drug alone. Our data suggest that the leupaxin-PTK2B axis plays an important role in acquired TKI resistance in AML. PTK2B/FAK inhibitors act synergistically with currently used therapeutics and may overcome emerging TKI resistance in FLT3-mutated AML at an early timepoint.

MicroRNA214 expression inhibits HCC cell proliferation through PTK2b/ Pyk2.

MicroRNAs (miRNAs/miRs) are crucial regulatory molecules that act as the most significantly downregulated microRNAs in hepatocellular carcinoma (HCC). PTK2b/Pyk2 is a non-receptor protein tyrosine kinase, which plays an important role in the development and metastasis of cancer. In this study, we explored the expression level and functional relationship between MicroRNA-214 (miR-214) and PTK2b/ Pyk2 in liver cancer cells. For this purpose, we analyzed the expression of miR-214 and PTK2b/Pyk2 in 38 cases of HCC and paired non-neoplastic tissue specimens using real-time PCR. MTT, cell cycle and construct recombinant plasmids analysis were used to explore the effects of miR-214 and PTK2b/Pyk2 on liver cancer cell proliferation. Results showed that the expression level of mir-214 in liver cancer tissues and liver cancer cell lines was significantly lower than that in normal tissues and cells, while the expression of PTK2b/Pyk2 was significantly increased. The overexpression of mir-214 or inhibition PTK2b/Pyk2 inhibited the proliferation of HCC cells. This research showed that mir-214 has an inhibitory effect on liver cancer through the expression of PTK2b/Pyk2.

A Novel Splicing Mutation c.335-1 G > A in the Cardiac Transcription Factor NKX2-5 Leads to Familial Atrial Septal Defect Through miR-19 and PYK2.

Mutations of NKX2-5 largely contribute to congenital heart diseases (CHDs), especially atrial septal defect (ASD). We identified a novel heterozygous splicing mutation c.335-1G > A in NKX2-5 gene in an ASD family via whole exome sequencing (WES) and linkage analysis. Utilizing the human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (hiPSC-CMs) as a disease model, we showed that haploinsufficiency of NKX2-5 contributed to aberrant orchestration of apoptosis and proliferation in ASD patient-derived hiPSC-CMs. RNA-seq profiling and dual-luciferase reporter assay revealed that NKX2-5 acts upstream of PYK2 via miR-19a and miR-19b (miR-19a/b) to regulate cardiomyocyte apoptosis. Meanwhile, miR-19a/b are also downstream mediators of NKX2-5 during cardiomyocyte proliferation. The novel splicing mutation c.335-1G > A in NKX2-5 and its potential pathogenic roles in ASD were demonstrated. Our work provides clues not only for deep understanding of NKX2-5 in cardia development, but also for better knowledge in the molecular mechanisms of CHDs.

Biliverdin reductase bridges focal adhesion kinase to Src to modulate synaptic signaling.

Synapses connect discrete neurons into vast networks that send, receive, and encode diverse forms of information. Synaptic function and plasticity, the neuronal process of adapting to diverse and variable inputs, depend on the dynamic nature of synaptic molecular components, which is mediated in part by cell adhesion signaling pathways. Here, we found that the enzyme biliverdin reductase (BVR) physically links together key focal adhesion signaling molecules at the synapse. BVR-null (BVR-/-) mice exhibited substantial deficits in learning and memory on neurocognitive tests, and hippocampal slices in which BVR was postsynaptically depleted showed deficits in electrophysiological responses to stimuli. RNA sequencing, biochemistry, and pathway analyses suggested that these deficits were mediated through the loss of focal adhesion signaling at both the transcriptional and biochemical level in the hippocampus. Independently of its catalytic function, BVR acted as a bridge between the primary focal adhesion signaling kinases FAK and Pyk2 and the effector kinase Src. Without BVR, FAK and Pyk2 did not bind to and stimulate Src, which then did not phosphorylate the N-methyl-d-aspartate (NMDA) receptor, a critical posttranslational modification for synaptic plasticity. Src itself is a molecular hub on which many signaling pathways converge to stimulate NMDAR-mediated neurotransmission, thus positioning BVR at a prominent intersection of synaptic signaling.

Alzheimer risk gene product Pyk2 suppresses tau phosphorylation and phenotypic effects of tauopathy.

BACKGROUND: Genetic variation at the PTK2B locus encoding the protein Pyk2 influences Alzheimer's disease risk. Neurons express Pyk2 and the protein is required for Amyloid-ß (Aß) peptide driven deficits of synaptic function and memory in mouse models, but Pyk2 deletion has minimal effect on neuro-inflammation. Previous in vitro data suggested that Pyk2 activity might enhance GSK3ß-dependent Tau phosphorylation and be required for tauopathy. Here, we examine the influence of Pyk2 on Tau phosphorylation and associated pathology. METHODS: The effect of Pyk2 on Tau phosphorylation was examined in cultured Hek cells through protein over-expression and in iPSC-derived human neurons through pharmacological Pyk2 inhibition. PS19 mice overexpressing the P301S mutant of human Tau were employed as an in vivo model of tauopathy. Phenotypes of PS19 mice with a targeted deletion of Pyk2 expression were compared with PS19 mice with intact Pyk2 expression. Phenotypes examined included Tau phosphorylation, Tau accumulation, synapse loss, gliosis, proteomic profiling and behavior. RESULTS: Over-expression experiments from Hek293T cells indicated that Pyk2 contributed to Tau phosphorylation, while iPSC-derived human neuronal cultures with endogenous protein levels supported the opposite conclusion. In vivo, multiple phenotypes of PS19 were exacerbated by Pyk2 deletion. In Pyk2-null PS19 mice, Tau phosphorylation and accumulation increased, mouse survival decreased, spatial memory was impaired and hippocampal C1q deposition increased relative to PS19 littermate controls. Proteomic profiles of Pyk2-null mouse brain revealed that several protein kinases known to interact with Tau are regulated by Pyk2. Endogenous Pyk2 suppresses LKB1 and p38 MAPK activity, validating one potential pathway contributing to increased Tau pathology. CONCLUSIONS: The absence of Pyk2 results in greater mutant Tau-dependent phenotypes in PS19 mice, in part via increased LKB1 and MAPK activity. These data suggest that in AD, while Pyk2 activity mediates Aß-driven deficits, Pyk2 suppresses Tau-related phenotypes.