PKN2 is essential for mouse embryonic development and proliferation of mouse fibroblasts

Danno, Sally; Kubouchi, Koji; Mehruba, Mona; Abe, Manabu; Natsume, Rie; Sakimura, Kenji; Eguchi, Satoshi; Oka, Masahiro; Hirashima, Masanori; Yasuda, Hiroki; Mukai, Hideyuki

doi:10.1111/gtc.12470

Cited by 18 publications

(19 citation statements)

References 66 publications

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“…PKN2 ‐edited clones were recovered but contained small deletions/insertions not affecting the open‐reading frames (see one example in ), suggesting that PKN2 is necessary for cell growth/survival of U937 cells. This observation is in line with the embryonic lethality of Pkn2 knock‐out in mice (Danno et al , ). We thus generated PKN1 KO clones expressing either WT or p.M694V Pyrin (Fig EV4A) and performed siRNA‐mediated knock‐down (KD) of PKN2 .…”

Section: Resultssupporting

confidence: 80%

Pyrin dephosphorylation is sufficient to trigger inflammasome activation in familial Mediterranean fever patients

et al. 2019

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Familial Mediterranean fever (FMF) is the most frequent hereditary systemic autoinflammatory syndrome. FMF is usually caused by biallelic mutations in the MEFV gene, encoding Pyrin. Conclusive genetic evidence lacks for about 30% of patients diagnosed with clinical FMF. Pyrin is an inflammasome sensor maintained inactive by two kinases (PKN1/2). The consequences of MEFV mutations on inflammasome activation are still poorly understood. Here, we demonstrate that PKC superfamily inhibitors trigger inflammasome activation in monocytes from FMF patients while they trigger a delayed apoptosis in monocytes from healthy donors. The expression of the pathogenic p.M694V MEFV allele is necessary and sufficient for PKC inhibitors (or mutations precluding Pyrin phosphorylation) to trigger caspase‐1‐ and gasdermin D‐mediated pyroptosis. In line with colchicine efficacy in patients, colchicine fully blocks this response in FMF patients’ monocytes. These results indicate that Pyrin inflammasome activation is solely controlled by Pyrin (de)phosphorylation in FMF patients while a second control mechanism restricts its activation in healthy donors/non‐FMF patients. This study paves the way toward a functional characterization of MEFV variants and a functional test to diagnose FMF.

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Section: Resultssupporting

confidence: 80%

Pyrin dephosphorylation is sufficient to trigger inflammasome activation in familial Mediterranean fever patients

et al. 2019

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“…RNA‐seq analysis of E9.0 embryos suggests that Pkn2 is more highly expressed than Pkn1 or Pkn3, and our analysis of lacZ reporter alleles for Pkn1 and Pkn2 confirms this view with Pkn2 showing widespread expression during early development. In agreement with previous work, it appears that Pkn2 null embryos are developmentally delayed by E7.5, but do establish proximo‐distal and anterior‐posterior axes, suggesting that embryonic patterning is relatively normal. However, axis extension and embryo turning are defective, consistent with cell motility defects as seen in vitro with Pkn depletion …”

Section: Discussionsupporting

confidence: 92%

The protein kinase C super‐family member PKN is regulated by mTOR and influences differentiation during prostate cancer progression

et al. 2017

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Background Phosphoinositide-3 (PI-3) kinase signaling has a pervasive role in cancer. One of the key effectors of PI-3 kinase signaling is AKT, a kinase that promotes growth and survival in a variety of cancers. Genetically engineered mouse models of prostate cancer have shown that AKT signaling is sufficient to induce prostatic epithelial neoplasia (PIN), but insufficient for progression to adenocarcinoma. This contrasts with the phenotype of mice with prostate-specific deletion of Pten, where excessive PI-3 kinase signaling induces both PIN and locally invasive carcinoma. We reasoned that additional PI-3 kinase effector kinases promote prostate cancer progression via activities that provide biological complementarity to AKT. We focused on the PKN kinase family members, which undergo activation in response to PI-3 kinase signaling, show expression changes in prostate cancer, and contribute to cell motility pathways in cancer cells. Methods PKN kinase activity was measured by incorporation of 32P into protein substrates. Phosphorylation of the turn-motif (TM) in PKN proteins by mTOR was analyzed using the TORC2-specific inhibitor torin and a PKN1 phospho-TM-specific antibody. Amino acid substitutions in the TM of PKN were engineered and assayed for effects on kinase activity. Cell motility-related functions and PKN localization was analyzed by depletion approaches and immunofluorescence microscopy, respectively. The contribution of PKN proteins to prostate tumorigenesis was characterized in several mouse models that express PKN transgenes. The requirement for PKN activity in prostate cancer initiated by loss of phosphatase and tensin homologue deleted on chromosome 10 (Pten), and the potential redundancy between PKN isoforms, was analyzed by prostate-specific deletion of Pkn1, Pkn2, and Pten. Results and Conclusions PKN1 and PKN2 contribute to motility pathways in human prostate cancer cells. PKN1 and PKN2 kinase activity is regulated by TORC2-dependent phosphorylation of the TM, which together with published data indicates that PKN proteins receive multiple PI-3 kinase-dependent inputs. Transgenic expression of active AKT and PKN1 is not sufficient for progression beyond PIN. Moreover, Pkn1 is not required for tumorigenesis initiated by loss of Pten. Triple knockout of Pten, Pkn1, and Pkn2 in mouse prostate results in squamous cell carcinoma, an uncommon but therapy-resistant form of prostate cancer.

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“…Immunoblot analysis was performed as previously described 81 . To preserve phosphorylated Akt protein, immediately after collection from mice under anesthesia, organs were frozen in liquid nitrogen, after which ice-cold TCA/acetone was applied to the frozen tissues for overnight incubation at −80 °C.…”

Section: Methodsmentioning

confidence: 99%

PKN1 kinase-negative knock-in mice develop splenomegaly and leukopenia at advanced age without obvious autoimmune-like phenotypes

Siddique

Kubouchi

Shinmichi

et al. 2019

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Protein kinase N1 (PKN1) knockout (KO) mice spontaneously form germinal centers (GCs) and develop an autoimmune-like disease with age. Here, we investigated the function of PKN1 kinase activity in vivo using aged mice deficient in kinase activity resulting from the introduction of a point mutation (T778A) in the activation loop of the enzyme. PKN1[T778A] mice reached adulthood without external abnormalities; however, the average spleen size and weight of aged PKN1[T778A] mice increased significantly compared to aged wild type (WT) mice. Histologic examination and Southern blot analyses of spleens showed extramedullary hematopoiesis and/or lymphomagenesis in some cases, although without significantly different incidences between PKN1[T778A] and WT mice. Additionally, flow cytometry revealed increased numbers in B220+, CD3+, Gr1+ and CD193+ leukocytes in the spleen of aged PKN1[T778A] mice, whereas the number of lymphocytes, neutrophils, eosinophils, and monocytes was reduced in the peripheral blood, suggesting an advanced impairment of leukocyte trafficking with age. Moreover, aged PKN1[T778A] mice showed no obvious GC formation nor autoimmune-like phenotypes, such as glomerulonephritis or increased anti-dsDNA antibody titer, in peripheral blood. Our results showing phenotypic differences between aged Pkn1-KO and PKN1[T778A] mice may provide insight into the importance of PKN1-specific kinase-independent functions in vivo.

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PKN2 is essential for mouse embryonic development and proliferation of mouse fibroblasts

Cited by 18 publications

References 66 publications

Pyrin dephosphorylation is sufficient to trigger inflammasome activation in familial Mediterranean fever patients

Pyrin dephosphorylation is sufficient to trigger inflammasome activation in familial Mediterranean fever patients

The protein kinase C super‐family member PKN is regulated by mTOR and influences differentiation during prostate cancer progression

PKN1 kinase-negative knock-in mice develop splenomegaly and leukopenia at advanced age without obvious autoimmune-like phenotypes

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