Intracellular C3 protects ß-cells from IL-1ß-driven cytotoxicity via interaction with Fyn-related kinase.

One of the hallmarks of type 1 but also type 2 diabetes is pancreatic islet inflammation, associated with altered pancreatic islet function and structure, if unresolved. IL-1ß is a proinflammatory cytokine which detrimentally affects ß-cell function. In the course of diabetes, complement components, including the central complement protein C3, are deregulated. Previously, we reported high C3 expression in human pancreatic islets, with upregulation after IL-1ß treatment. In the current investigation, using primary human and rodent material and CRISPR/Cas9 gene-edited ß-cells deficient in C3, or producing only cytosolic C3 from a noncanonical in-frame start codon, we report a protective effect of C3 against IL-1ß-induced ß-cell death, that is attributed to the cytosolic fraction of C3. Further investigation revealed that intracellular C3 alleviates IL-1ß-induced ß-cell death, by interaction with and inhibition of Fyn-related kinase (FRK), which is involved in the response of ß-cells to cytokines. Furthermore, these data were supported by increased ß-cell death in vivo in a ß-cell-specific C3 knockout mouse. Our data indicate that a functional, cytoprotective association exists between FRK and cytosolic C3.

Hemifacial microsomia is linked to a rare homozygous variant V162I in FRK and validated in zebrafish.

OBJECTIVES: Hemifacial microsomia (HFM) is a common birth defect involving the first and second branchial arch derivatives. Although several chromosomal abnormalities and causal gene variants have been identified, genetic etiologies in a majority of cases with HFM remain unknown. This study aimed to identify genetic mutations in affected individuals with HFM. METHODS: Whole-exome sequencing and bioinformatics analysis were performed for 16 affected individuals and their family members. Sanger sequencing was applied for confirmation of selected mutations. Zebrafish embryos were used for in situ hybridization of candidate gene, microinjection with antisense morpholino, and cartilage staining. RESULTS: A homozygous missense mutation (c.484G > A; p.V162I) in the FRK gene was identified in an 18-year-old girl with HFM and dental abnormalities. Heterozygous mutation of this mutation was identified in her parents, who are first cousins in a consanguineous family. FRK is highly expressed in the Meckel's cartilage during embryonic development in mouse and zebrafish. Knockdown of frk in zebrafish showed a lower length and width ratio of Meckel's cartilage, abnormal mandibular jaw joint, and disorganized ceratobranchial cartilage and bone. CONCLUSIONS: We identified a recessive variant in the FRK gene as a novel candidate gene for a patient with HFM and mandibular hypoplasia and revealed its effects on craniofacial and embryonic development in zebrafish.

A Comparative Characterization and Expression Profiling Analysis of Fructokinase and Fructokinase-like Genes: Exploring Their Roles in Cucumber Development and Chlorophyll Biosynthesis.

Fructokinase (FRK) and fructokinase-like (FLN), belonging to the phosphofructokinase B type subfamily, share substantial sequence similarity, and are crucial in various plant physiological processes. However, there is limited information regarding what functionally differentiates plant FRKs from FLNs. Here, a total of three CsFRKs and two CsFLNs were identified from the cucumber genome. Their significant difference lay in the structure of their G/AXGD motif, which existed as GAGD in CsFRKs, but as G/ASGD in CsFLNs. Comparative phylogenetic analysis classified CsFRKs and CsFLNs into five sub-branches consistent with their quite different exon/intron organizations. Both transcriptome data and RT-qPCR analyses revealed that CsFRK3 was the most active gene, with the highest expression in the majority of tissues tested. Moreover, the expression levels of two putative plastidic genes, CsFRK1 and CsFLN2, were significantly positively associated with chlorophyll accumulation in the chlorophyll-reduced cucumber mutant. Briefly, both CsFRK and CsFLN genes were involved in the development of sink tissues, especially CsFRK3. CsFRK1 and CsFLN2 were recognized as candidates in the chlorophyll biosynthesis pathway of cucumber. These results would greatly assist in further investigation on functional characterization of FRKs and FLNs, especially in the development and chlorophyll biosynthesis of cucumber.

LncRNA HOTTIP leads to osteoarthritis progression via regulating miR-663a/ Fyn-related kinase axis.

BACKGROUND: Long non-coding RNA (lncRNA) has been implicated in the progression of osteoarthritis (OA). This study was aimed to explore the role and molecular mechanism of lncRNA HOXA terminal transcriptional RNA (HOTTIP) in the development of OA. METHODS: The expression of HOTTIP, miR-663a and Fyn-related kinase (FRK) in the OA articular cartilage and OA chondrocyte model induced by IL-1ß was determined by qRT-PCR. CCK-8, colony formation and flow cytometry were used to determine the cell proliferation and apoptosis of OA chondrocytes. The specific molecular mechanism of HOTTIP in OA chondrocytes was determined by dual luciferase reporter assay, qRT-PCR, western blotting and RNA pull-down. RESULTS: The expression of HOTTIP and FRK were up-regulated, while miR-663a was down-regulated in OA cartilage tissues. Knockdown of HOTTIP decreased the proliferation and induced the apoptosis of OA cartilage model cells, while overexpression of HOTTIP increased the proliferation and reduced the apoptosis of OA cartilage model cells. Moreover, HOTTIP could bind to miR-663a as competitive endogenous RNA. Inhibition of miR-663a expression could alleviate the effect of HOTTIP knockdown on the proliferation and apoptosis of OA cartilage model cells. Furthermore, FRK was found to be a direct target of miR-663a, which could markedly down-regulate the expression of FRK in OA chondrocytes, while HOTTIP could remarkably up-regulate the expression of FRK. In addition, miR-663a inhibition increased the proliferation and reduced the apoptosis of OA cells, while FRK knockdown reversed the effect of miR-663a inhibition on the proliferation and apoptosis of OA cells. Meanwhile, overexpression of miR-663a decreased the proliferation and induced the apoptosis of OA cells, while overexpression of FRK reversed the effect of miR-663a overexpression on the proliferation and apoptosis of OA cells. CONCLUSION: HOTTIP was involved in the proliferation and apoptosis of OA chondrocytes via miR-663a/ FRK axis, and HOTTIP/miR-663a/FRK might be a potential target for the treatment of OA.

FRK plays an oncogenic role in non-small cell lung cancer by enhancing the stemness phenotype via induction of metabolic reprogramming.

The role of Fyn-related kinase (FRK) in malignant tumors remains controversial. Our study investigated the function of FRK in lung cancer. Immunohistochemistry staining and generating a knockout of FRK by CRISPR/Cas9 in H1299 (FRK-KO-H1299) cells were strategies used to explore the role of FRK. Immunohistochemistry staining indicated that FRK expression was elevated in 223 lung cancer tissues compared to 26 distant normal lung tissues. FRK contributed to poor survival status in lung cancer patients and acted as a predictor for poor prognosis of lung cancer. Knockout of FRK by CRISPR/Cas9 markedly inhibited proliferation, invasion, colony formation and epithelial-mesenchymal transition (EMT) process in the lung cancer cell line H1299. Further exploration indicated that FRK-KO damaged the stemness phenotype of H1299 by inhibiting CD44 and CD133 expression. Seahorse detection and a U-13 C flux assay revealed that FRK-KO induced metabolism reprogramming by inhibiting the Warburg effect and changing the energy type in H1299 cells. Epidermal growth factor stimulation recovered the expression of FRK and biological functions, metabolic reprogramming and stemness phenotype of H1299 cells. FRK plays an oncogenic role in lung cancer cells via a novel regulation mechanism of enhancing the stemness of H1299 cells by inducing metabolism reprogramming, which finally promotes EMT and metastasis. Our study also indicates that FRK could be used as a potential therapeutic target for drug development.

TRIM44 promotes cell proliferation and migration by inhibiting FRK in renal cell carcinoma.

TRIM44 has oncogenic roles in various cancers. However, TRIM44 expression and its function in renal cell carcinoma (RCC) are still unknown. Here in this study, we investigated the clinical significance of TRIM44 and its biological function in RCC. TRIM44 overexpression was significantly associated with clinical M stage, histologic type (clear cell) and presence of lymphatic invasion (P = .047, P = .005, and P = .028, respectively). Moreover, TRIM44 overexpression was significantly associated with poor prognosis in terms of cancer-specific survival (P = .019). Gain-of-function and loss-of-function studies using TRIM44 and siTRIM44 transfection showed that TRIM44 promotes cell proliferation and cell migration in two RCC cell lines, Caki1 and 769P. To further investigate the role of TRIM44 in RCC, we performed integrated microarray analysis in Caki1 and 769P cells and explored the data in the Oncomine database. Interestingly, FRK was identified as a promising candidate target gene of TRIM44, which was downregulated in RCC compared with normal renal tissues. We found that cell proliferation was inhibited by TRIM44 knockdown and then recovered by siFRK treatment. Taken together, the present study revealed the association between high expression of TRIM44 and poor prognosis in RCC patients and that TRIM44 promotes cell proliferation by regulating FRK.

Down-Regulation of miR-2053 Inhibits the Development and Progression of Esophageal Carcinoma by Targeting Fyn-Related Kinase (FRK).

BACKGROUND: MicroRNAs (miRNAs) play essential roles in the regulation and pathophysiology of various types of cancers including esophageal carcinoma (ESCA). Increasing numbers of miRNAs have been identified to be important regulators in the progression of ESCA by regulating gene expression. However, functional miRNAs and the underlying mechanisms involved in ESCA need sufficient elucidation. AIMS: In the present study, the function of miR-2053 was investigated in ESCA cells. METHODS: The expression of miR-2053 was detected in four different ESCA cell lines (Eca109, Ec9706, KYSE30, and TE-1 cells) and normal cell line (HEEC) by qRT-PCR. Cell proliferation, migration, and invasion abilities after knockdown of miR-2053 were assessed by CCK-8 assay, scratch assay, and transwell assay, respectively. Cell cycle of ESCA cells was detected by flow cytometric analysis. Expression of proteins in ESCA cells was detected by Western blot analysis. RESULTS: The results showed that the expression of miR-2053 was remarkably up-regulated in ESCA tissues and cells lines. Down-regulation of miR-2053 markedly inhibited cell proliferation, migration, and invasion and markedly induced cell cycle arrest and cell apoptosis in ESCA cell lines. Fyn-related kinase (FRK) was a target gene of miR-2053. Moreover, down-regulation of miR-2053 mediated the protein kinase B (AKT)/mammalian target of rapamycin and Wnt3a/ß-catenin signaling pathway in ESCA cell lines. CONCLUSIONS: Our results together suggest the potential of regulating miR-2053 expression against development and progression of esophageal carcinoma by targeting FRK.

FRK suppresses human glioma growth by inhibiting ITGB1/FAK signaling.

Fyn-related kinase (FRK), a member of the Src-related tyrosine kinase family, functions as a tumor suppressor in several malignancies. We previously showed that FRK overexpression inhibited the growth of glioma cells. However, it is unknown whether FRK is equally effective against intracranial glioma in vivo, and the mechanism by which FRK influences glioma cell growth remains unclear. In this study, we found that tumor volume was reduced by about one-third in mice with FRK overexpression, which showed improved survival relative to controls. Immunofluorescence analysis revealed that FRK overexpression inhibited glioma cell proliferation and induced their apoptosis. Importantly, in vitro we further found that FRK decreased the expression of integrin subunit ß1 (ITGB1) at both the mRNA and protein levels. FRK also inhibited transactivation by ITGB1, resulting in the suppression of its target proteins AKT and focal adhesion kinase (FAK). ITGB1 overexpression promoted glioma cell growth and partially reduced FRK-induced growth suppression. These results indicate that FRK inhibits human glioma growth via regulating ITGB1/FAK signaling and provide a potential therapeutic target for the treatment of glioma.

Activation of STAT1 by the FRK tyrosine kinase is associated with human glioma growth.

PURPOSE: Glioma is a highly aggressive and lethal brain tumor. Signal transducers and activators of transcription (STAT) pathway are widely implicated in glioma carcinogenesis. Our previous study found that the Fynrelated kinase (FRK) gene, plays as a tumor suppressor in the development and progression of glioma. This study aimed to investigate the role of FRK in the activation pathway of STATs and its effect on the growth of glioma. METHODS: The U251 and U87 cells with stable FRK overexpression were generated by lentivirus technique. The effects of FRK on the related proteins of STAT signaling pathway were detected by western blotting. Coimmunoprecipitation was used to detect the association of FRK and STAT1. The effects of STAT1 on the proliferation of glioma cells were detected by CCK8 or Edu cell proliferation assays. The expressions and correlation of FRK and p-STAT1 in glioma tissues were detectd by western blotting or immunohistochemistry. The effect of FRK on the growth of glioma was investigated in vivo mouse model. RESULTS: The level of p-JAK2 and p-STAT1 increased after FRK overexpression, while they decreased after FRK downregulation both in U251 and U87 cells. However, FRK had no effect on STAT3 phosphorylation. FRK-induced STAT1 activation was not dependent on JAK2. FRK associated with STAT1, induced STAT1 nuclear translocation and regulated the expressions of STAT1-related target genes. STAT1 overexpression suppressed the proliferation of glioma cells. In contrast, STAT1 knockdown by siRNA promoted glioma cell growth. Importantly, down-regulation of STAT1 partially attenuated FRK-induced growth suppression. The clinical sample-based study indicated that the expression of FRK was significantly correlated with the expression of p-STAT1. FRK significantly inhibited glioma tumor growth in vivo. CONCLUSIONS: Our findings highlighted a critical role of FRK in tumor suppression ability through promoting STAT1 activation, and provided a potential therapeutic target for glioma.

Global phosphoproteomic analysis identifies SRMS-regulated secondary signaling intermediates.

BACKGROUND: The non-receptor tyrosine kinase, SRMS (Src-related kinase lacking C-terminal regulatory tyrosine and N-terminal myristoylation sites) is a member of the BRK family kinases (BFKs) which represents an evolutionarily conserved relative of the Src family kinases (SFKs). Tyrosine kinases are known to regulate a number of cellular processes and pathways via phosphorylating substrate proteins directly and/or by partaking in signaling cross-talks leading to the indirect modulation of various signaling intermediates. In a previous study, we profiled the tyrosine-phosphoproteome of SRMS and identified multiple candidate substrates of the kinase. The broader cellular signaling intermediates of SRMS are unknown. METHODS: In order to uncover the broader SRMS-regulated phosphoproteome and identify the SRMS-regulated indirect signaling intermediates, we performed label-free global phosphoproteomics analysis on cells expressing wild-type SRMS. Using computational database searching and bioinformatics analyses we characterized the dataset. RESULTS: Our analyses identified 60 hyperphosphorylated (phosphoserine/phosphothreonine) proteins mapped from 140 hyperphosphorylated peptides. Bioinfomatics analyses identified a number of significantly enriched biological and cellular processes among which DNA repair pathways were found to be upregulated while apoptotic pathways were found to be downregulated. Analyses of motifs derived from the upregulated phosphosites identified Casein kinase 2 alpha (CK2α) as one of the major potential kinases contributing to the SRMS-dependent indirect regulation of signaling intermediates. CONCLUSIONS: Overall, our phosphoproteomics analyses identified serine/threonine phosphorylation dynamics as important secondary events of the SRMS-regulated phosphoproteome with implications in the regulation of cellular and biological processes.

Understanding the cellular roles of Fyn-related kinase (FRK): implications in cancer biology.

The non-receptor tyrosine kinase Fyn-related kinase (FRK) is a member of the BRK family kinases (BFKs) and is distantly related to the Src family kinases (SFKs). FRK was first discovered in 1993, and studies pursued thereafter attributed a potential tumour-suppressive function to the enzyme. In recent years, however, further functional characterization of the tyrosine kinase in diverse cancer types suggests that FRK may potentially play an oncogenic role as well. Specifically, while ectopic expression of FRK suppresses cell proliferation and migration in breast and brain cancers, knockdown or catalytic inhibition of FRK suppresses these cellular processes in pancreatic and liver cancer. Such functional paradox is therefore evidently exhibited in a tissue-specific context. This review sheds light on the recent developments emerged from investigations on FRK which include: (a) a review of the expression pattern of the protein in mammalian cells/tissues, (b) underlying genomic perturbations and (c) a mechanistic function of the enzyme across different cellular environments. Given its functional heterogeneity observed across different cancers, we also discuss the therapeutic significance of FRK.

Leukaemic cells expressing ETV6::FRK are sensitive to dasatinib in vivo.

ETV6::Fyn-related kinase (FRK), which is a Src family tyrosine-kinase-related fusion gene and firstly identified in our patient with paediatric high risk B cell precursor acute lymphoblastic leukaemia (B-ALL), has no evidence of efficacy of tyrosine kinase inhibitor in vivo. We performed functional analysis of ETV6::FRK to establish molecular targeting therapy and determined that dasatinib could abrogate proliferation activity of ETV6::FRK through the repression of FRK-STAT3/STAT5 pathway in vitro and significantly extended the survival time of the xenografted mice in vivo (p < 0.01). Our data support the potential of dasatinib as a therapeutic option for patients with B-ALL harboring FRK rearrangements.

Seeking a better understanding of the non-receptor tyrosine kinase, SRMS.

SRMS (Src-Related kinase lacking C-terminal regulatory tyrosine and N-terminal Myristoylation Sites) is a non-receptor tyrosine kinase first reported in a 1994 screen for genes regulating murine neural precursor cells. SRMS, pronounced "Shrims", lacks the C-terminal regulatory tyrosine critical for the regulation of the enzymatic activity of Src-family kinases (SFKs). Another remarkable characteristic of SRMS is its localization into distinct SRMS cytoplasmic punctae (SCPs) or GREL (Goel Raghuveera-Erique Lukong) bodies, a pattern not observed in the SFKs. This unique subcellular localization of SRMS could dictate its cellular targets, proteome, and potentially, substrates. However, the function of SRMS is still relatively unknown. Further, how is its activity regulated and by what cellular targets? Studies have emerged highlighting the potential role of SRMS in autophagy and in regulating the activation of BRK/PTK6. Potential novel cellular substrates have also been identified, including DOK1, vimentin, Sam68, FBKP51, and OTUB1. Recent studies have also demonstrated the potential role of the kinase in various cancers, including gastric and colorectal cancers and platinum resistance in ovarian cancer. This review discusses the advancements made in SRMS-related biology to date and the path to understanding the cellular and physiological significance of the kinase.

HIV-1 Vif suppresses antiviral immunity by targeting STING.

HIV-1 infection-induced cGAS-STING-TBK1-IRF3 signaling activates innate immunity to produce type I interferon (IFN). The HIV-1 nonstructural protein viral infectivity factor (Vif) is essential in HIV-1 replication, as it degrades the host restriction factor APOBEC3G. However, whether and how it regulates the host immune response remains to be determined. In this study, we found that Vif inhibited the production of type I IFN to promote immune evasion. HIV-1 infection induced the activation of the host tyrosine kinase FRK, which subsequently phosphorylated the immunoreceptor tyrosine-based inhibitory motif (ITIM) of Vif and enhanced the interaction between Vif and the cellular tyrosine phosphatase SHP-1 to inhibit type I IFN. Mechanistically, the association of Vif with SHP-1 facilitated SHP-1 recruitment to STING and inhibited the K63-linked ubiquitination of STING at Lys337 by dephosphorylating STING at Tyr162. However, the FRK inhibitor D-65495 counteracted the phosphorylation of Vif to block the immune evasion of HIV-1 and antagonize infection. These findings reveal a previously unknown mechanism through which HIV-1 evades antiviral immunity via the ITIM-containing protein to inhibit the posttranslational modification of STING. These results provide a molecular basis for the development of new therapeutic strategies to treat HIV-1 infection.

Impact of supplemented undenatured type II collagen on pain and mobility in healthy Labrador Retrievers during an exercise regimen.

The aim of this experiment was to evaluate the effect of undenatured type II collagen supplementation on inflammation and pain using gait analysis and industry-accepted pain and mobility questionnaires during an exercise regimen in healthy dogs. Forty healthy Labrador Retrievers (20 male/20 female; range: 5 to 12 yr) were sorted into two groups: undenatured type II collagen group receiving 40 mg UC-II product (10 mg total collagen and ≥3% undenatured type II collagen) and placebo group receiving 40 mg maltodextrin daily by capsule. After 2 wk loading, all dogs began an 11 wk endurance exercise regimen consisting of two weekly runs, starting at 5 km and increasingly incrementally to 8 km, with one final 16 km run. Gait analysis was performed at baseline; before, 24 and 48 h after the first 5 km run; and before, 24 and 48 h after the final 16 km run. Gait analysis was calculated to obtain a Four Rivers Kennel (FRK) Inflammation Index score. Dogs were scored according to the Liverpool Osteoarthritis in Dogs (LOAD) and Canine Brief Pain Inventory (CBPI) assessments at baseline, before and after the first 5 km run, and before and after the final 16 km run. On the LOAD questionnaire, undenatured type II collagen group had improved "how active is the dog" (P = 0.03) and less "stiffness after a lie down" (P = 0.041) compared with placebo at pre 5 km. Undenatured type II collagen appeared to mitigate the development of pain after exercise compared with placebo, as related to the CPBI assessment. Undenatured type II collagen dogs had lower "pain at worst" pre 5 km (P = 0.021), "pain at least" post 5 km (P = 0.015), "pain at average" post 5 km (P = 0.046), and "pain as it is now" post 16 km (P = 0.006) compared with placebo dogs. Undenatured type II collagen was more effective than placebo at mitigating inflammation on gait analysis per the FRK Inflammation Index. Undenatured type II collagen dogs had a 6.42 lower FRK Inflammation Index score at 24 h post 5 km (P = 0.032) and 6.3 lower score at 24 h post 16 km (P = 0.029), indicating the mitigation of inflammation on gait analysis. When considering the change between timepoints, undenatured type II collagen had a lower increase in FRK Inflammation scores compared with placebo for baseline to pre 5 km (P < 0.001), pre 16 km to 24 h post 16 km (P = 0.028), and pre 16 km to 48 h post 16 km (P = 0.027). Undenatured type II collagen supplemented Labrador Retrievers improved pain assessment variables and improved FRK Inflammation Index on gait analysis.

FRK inhibits glioblastoma progression via phosphorylating YAP and inducing its ubiquitylation and degradation by Siah1.

BACKGROUND: We previously report that yes-associated protein (YAP), the core downstream effector of Hippo pathway, promotes the malignant progression of glioblastoma (GBM). However, although classical regulatory mechanisms of YAP are well explored, how YAP is modulated by the Hippo-independent manner remains poorly understood. Meanwhile, the nonreceptor tyrosine kinase Fyn-related kinase (FRK), which exhibits low expression and possesses tumor suppressor effects in GBM, is reported to be involved in regulation of protein phosphorylation. Here, we examined whether FRK could impede tumor progression by modulating YAP activities. METHODS: Human GBM cells and intracranial GBM model were used to assess the effects of FRK and YAP on the malignant biological behaviors of GBM. Immunoblotting and immunohistochemistry were used to detect the expression of core proteins in GBM tissues. Co-immunoprecipitation, proximity ligation assay, luciferase assay and ubiquitination assay were utilized to determine the protein-protein interactions and related molecular mechanisms. RESULTS: The expression levels of FRK and YAP were inversely correlated with each other in glioma tissues. In addition, FRK promoted the ubiquitination and degradation of YAP, leading to tumor suppression in vitro and in vivo. Mechanistically, FRK interacted with and phosphorylated YAP on Tyr391/407/444, which recruited the classical E3 ubiquitin ligase Siah1 to catalyze ubiquitination and eventually degradation of YAP. Siah1 is required for YAP destabilization initiated by FRK. CONCLUSIONS: We identify a novel mechanism by which FRK orchestrates tumor-suppression effect through phosphorylating YAP and inducing its ubiquitination by Siah1. FRK-Siah1-YAP signaling axis may serve as a potential therapeutic target for GBM treatment.

Identification and relative expression analysis of CaFRK gene family in pepper.

Fructokinase is the main catalytic enzyme for fructose phosphorylation and can also act as a glucose receptor and signal molecule to regulate the metabolism of plants, which plays an important role in plant growth and development. In this study, the CaFRK gene family and their molecular characteristics are systematically identified and analyzed, and the specific expression of CaFRKs under different tissues, abiotic stresses and hormone treatments were explored. Nine FRK genes were authenticated in pepper genome database, which were dispersedly distributed on eight reference chromosomes and predicted to localize in the cytoplasm. Many cis-acting elements that respond to light, different stresses, hormones and tissue-specific expression were found in the promoters of CaFRKs. FRK proteins of four species including Capsicum annuum, Arabidopsis thaliana, Solanum lycopersicum and Oryza sativa were divided into four groups via phylogenetic analysis. The collinearity analysis showed that there were two collinear gene pairs between CaFRKs and AtFRKs. In addition, it was significantly found that CaFRK9 expressed far higher in flower than other tissues, and the relative expression of CaFRK9 was gradually enhanced with the development of flower buds in fertile accessions, 8B, R1 and F1. Nevertheless, CaFRK9 hardly expressed in all stages of cytoplasmic male sterile lines. Based on the quantitative real-time PCR, most of CaFRK genes showed significant up-regulation under low-temperature, NaCl and PEG6000 treatments. On the contrary, the expression levels of most CaFRKs revealed a various trend in response to hormone treatments (IAA, ABA, GA3, SA and MeJA). This study systematically analyzed CaFRK gene family and studied its expression pattern, which lay the foundation of CaFRK genes cloning and functional verification response to abiotic stresses, and provides new insights into exploring the CaFRK genes on the pollen development in pepper. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03196-1.

Evolution and Functional Divergence of the Fructokinase Gene Family in Populus.

New kinase has emerged throughout evolution, but how new kinase evolve while maintaining their functions and acquiring new functions remains unclear. Fructokinase (FRK), the gateway kinase to fructose metabolism, plays essential roles in plant development, and stress tolerance. Here, we explored the evolution of FRK gene family in 20 plant species (from green algae to angiosperms) and their functional roles in Populus. We identified 125 putative FRK genes in the 20 plant species with an average of 6 members per species. Phylogenetic analysis separated these 125 genes into 8 clades including 3 conserved clades and 5 specific clades, the 5 of which only exist in green algae or angiosperms. Evolutionary analysis revealed that FRK genes in ancient land plants have the largest number of functional domains with the longest amino acid sequences, and the length of FRK genes became shorter during the transition to vascular plants. This was accompanied by loss, acquisition, and diversification of functional domains. In Populus, segmental duplication appears to be the main mechanism for the expansion of FRK genes. Specially, most FRK genes duplicated in salicoids are regulated by Populus-specific microRNAs. Furthermore, compared with common FRKs, Populus-specific FRKs have showed higher expression specificity and are associated with fewer growth and wood property traits, which suggests that these FRKs may have undergone functional divergence. Our study explores the specific roles of FRKs in the Populus genome and provides new insights for functional investigation of this gene family.

miR-19 promotes the proliferation of clear cell renal cell carcinoma by targeting the FRK-PTEN axis.

BACKGROUND: The non-receptor tyrosine kinase Fyn-related kinase (FRK) has been reported to affect cell proliferation in several cancer types. However, its effect on the proliferation of clear cell renal cell carcinoma (ccRCC) remains largely unknown. PURPOSE: The objective of this study was to investigate the expression pattern and function of FRK in ccRCC. We further determined how FRK interacted with other molecules to regulate ccRCC proliferation. PATIENTS AND METHODS: The expression of FRK in ccRCC samples and paired normal renal tissues from 30 patients were analyzed by immunoblotting, immunohistochemistry and quantitative PCR. Then the role of FRK in ccRCC proliferation was analyzed by Cell Counting Kit-8, colony formation assay and EdU incorporation assay. In addition, the miRNA targeting FRK was predicted through a bioinformatic approach and validated by quantitative PCR, immunoblotting and luciferase reporter assay. Finally, the underlying mechanism of FRK regulation of ccRCC proliferation was also determined. RESULTS: Low expression of FRK was detected in ccRCC samples and predicted poor survival for ccRCC patients. FRK inhibited the proliferation of ccRCC cells via phosphorylating downstream PTEN. miR-19 was identified as a novel suppressor of FRK in renal cancer cells and it promoted the proliferation of ccRCC by inhibiting the FRK-PTEN axis. CONCLUSION: Our results unravel a new regulatory mechanism involved in ccRCC proliferation and may be useful in the identification of therapeutic targets for ccRCC.

A new insight into the evolution and functional divergence of FRK genes in Pyrus bretschneideri.

In plants, plant fructokinases (FRKs) are considered to be the main gateway of fructose metabolism as they can phosphorylate fructose to fructose-6-phosphate. Chinese white pears (Pyrus bretschneideri) are one of the popular fruits in the world market; sugar content is an important factor affecting the quality of the fruit. We identified 49 FRKs from four Rosaceae species; 20 of these sequences were from Chinese white pear. Subsequently, phylogenic relationship, gene structure and micro-collinearity were analysed. Phylogenetic and exon-intron analysis classified these FRKs into 10 subfamilies, and it was aimed to further reveal the variation of the gene structure and the evolutionary relationship of this gene family. Remarkably, gene expression patterns in different tissues or different development stages of the pear fruit suggested functional redundancy for PbFRKs derived from segmental duplication or genome-wide duplication and sub-functionalization for some of them. Additionally, PbFRK11, PbFRK13 and PbFRK16 were found to play important roles in regulating the sugar content in the fruit. Overall, this study provided important insights into the evolution of the FRK gene family in four Rosaceae species, and highlighted its roles in both pear tissue and fruits. Results presented here provide the appropriate candidate of PbFRKs that might contribute to fructose efflux in the pear fruit.