RESUMEN
PKN2, a member of the protein kinase N (PKN) family, has been suggested by in vitro culture cell experiments to bind to Rho/Rac GTPases and contributes to cell-cell contact and cell migration. To unravel the in vivo physiological function of PKN2, we targeted the PKN2 gene. Constitutive disruption of the mouse PKN2 gene resulted in growth retardation and lethality before embryonic day (E) 10.5. PKN2-/- embryo did not undergo axial turning and showed insufficient closure of the neural tube. Mouse embryonic fibroblasts (MEFs) derived from PKN2-/- embryos at E9.5 failed to grow. Cre-mediated ablation of PKN2 in PKN2flox/flox MEFs obtained from E14.5 embryos showed impaired cell proliferation, and cell cycle analysis of these MEFs showed a decrease in S-phase population. Our results show that PKN2 is essential for mouse embryonic development and cell-autonomous proliferation of primary MEFs in culture. Comparison of the PKN2-/- phenotype with the phenotypes of PKN1 and PKN3 knockout strains suggests that PKN2 has distinct nonredundant functions in vivo, despite the structural similarity and evolutionary relationship among the three isoforms.
Asunto(s)
Desarrollo Embrionario/fisiología , Fibroblastos/citología , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismo , Animales , Movimiento Celular/fisiología , Proliferación Celular/fisiología , Embrión de Mamíferos/citología , Desarrollo Embrionario/genética , Femenino , Fibroblastos/metabolismo , Ratones , Ratones Noqueados , Fenotipo , EmbarazoRESUMEN
The role of Protein Kinase N2 (PKN2, also known as PRK2/PKNγ) in cell aggregate/spheroid formation in suspension culture was investigated using immortalized fibroblasts established from PKN2 flox/flox mouse embryos. PKN2 flox/flox cells formed cell aggregates in flat bottom low attachment well plates, such as 2% agar and poly-2-hydroxyethymethacrylate coated plates, however, Cre;PKN2 flox/flox cells in which PKN2 was depleted by the introduction of Cre-recombinase rarely formed aggregates. Time-lapse analysis revealed that the velocity of Cre;PKN2 flox/flox cell motility was significantly lower than that of PKN2 flox/flox in a low attachment flat-bottom plate, which likely resulted in a lower cell-cell contact frequency among Cre;PKN2 flox/flox cells. Conversely, Cre;PKN2 flox/flox cells could form initial cell aggregates in U-bottom low attachment well plates, however, the succeeding compaction process was delayed in Cre;PKN2 flox/flox cells with decreased roundness, although PKN2 flox/flox cells underwent compaction in a round shape spheroid within 24 h. Immunoblot analysis revealed that the preparation of the cell suspension from adherent conditions using trypsin/EDTA treatment significantly decreased the expression of N-cadherin in both PKN2 flox/flox and Cre;PKN2 flox/flox cells. The N-cadherin expression level recovered time-dependently; however, the recovery of N-cadherin was significantly delayed in Cre;PKN2 flox/flox cells compared to PKN2 flox/flox cells. Reverse transcription quantitative PCR revealed that N-cadherin mRNA in Cre;PKN2 flox/flox cells was significantly lower than that of PKN2 flox/flox cells. These results suggest that PKN2 controls the velocity of cell motility and the transcription of N-cadherin in fibroblasts, leading to cell aggregation and compaction for spheroid formation in suspension culture.
RESUMEN
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.
Asunto(s)
Leucopenia/genética , Proteína Quinasa C/genética , Esplenomegalia/genética , Factores de Edad , Animales , Técnicas de Sustitución del Gen , Centro Germinal/metabolismo , Leucopenia/metabolismo , Ratones , Ratones Noqueados , Fosforilación , Proteína Quinasa C/metabolismo , Transducción de Señal/fisiología , Esplenomegalia/metabolismoRESUMEN
Knock-in mice lacking PKN1 kinase activity were generated by introducing a T778A point mutation in the catalytic domain. PKN1[T778A] mutant mice developed to adulthood without apparent external abnormalities, but exhibited lower T and B lymphocyte counts in the peripheral blood than those of wild-type (WT) mice. T and B cell development proceeded in an apparently normal fashion in bone marrow and thymus of PKN1[T778A] mice, however, the number of T and B cell counts were significantly higher in the lymph nodes and spleen of mutant mice in those of WT mice. After transfusion into WT recipients, EGFP-labelled PKN1[T778A] donor lymphocytes were significantly less abundant in the peripheral circulation and more abundant in the spleen and lymph nodes of recipient mice compared with EGFP-labelled WT donor lymphocytes, likely reflecting lymphocyte sequestration in the spleen and lymph nodes in a cell-autonomous fashion. PKN1[T778A] lymphocytes showed significantly lower chemotaxis towards chemokines and sphingosine 1-phosphate (S1P) than WT cells in vitro. The biggest migration defect was observed in response to S1P, which is essential for lymphocyte egress from secondary lymphoid organs. These results reveal a novel role of PKN1 in lymphocyte migration and localization.
Asunto(s)
Quimiotaxis de Leucocito/genética , Quimiotaxis de Leucocito/inmunología , Linfocitos/inmunología , Linfocitos/metabolismo , Proteína Quinasa C/deficiencia , Animales , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Quimiocinas/metabolismo , Sitios Genéticos , Genotipo , Inmunofenotipificación , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Recuento de Linfocitos , Linfocitos/citología , Lisofosfolípidos/metabolismo , Ratones , Ratones Transgénicos , Esfingosina/análogos & derivados , Esfingosina/metabolismoRESUMEN
PKN, a conserved family member related to PKC, was the first protein kinase identified as a target of the small GTPase Rho. PKN is involved in various functions including cytoskeletal arrangement and cell adhesion. Furthermore, the enrichment of PKN3 mRNA in some cancer cell lines as well as its requirement in malignant prostate cell growth suggested its involvement in oncogenesis. Despite intensive research efforts, physiological as well as pathological roles of PKN3 in vivo remain elusive. Here, we generated mice with a targeted deletion of PKN3. The PKN3 knockout (KO) mice are viable and develop normally. However, the absence of PKN3 had an impact on angiogenesis as evidenced by marked suppressions of micro-vessel sprouting in ex vivo aortic ring assay and in vivo corneal pocket assay. Furthermore, the PKN3 KO mice exhibited an impaired lung metastasis of melanoma cells when administered from the tail vein. Importantly, PKN3 knock-down by small interfering RNA (siRNA) induced a glycosylation defect of cell-surface glycoproteins, including ICAM-1, integrin ß1 and integrin α5 in HUVECs. Our data provide the first in vivo genetic demonstration that PKN3 plays critical roles in angiogenesis and tumor metastasis, and that defective maturation of cell surface glycoproteins might underlie these phenotypes.
Asunto(s)
Carcinoma Pulmonar de Lewis/genética , Regulación Neoplásica de la Expresión Génica , Melanoma Experimental/genética , Neovascularización Patológica/genética , Neovascularización Fisiológica/genética , Proteína Quinasa C/genética , Animales , Aorta/metabolismo , Aorta/patología , Bioensayo , Carcinoma Pulmonar de Lewis/metabolismo , Carcinoma Pulmonar de Lewis/patología , Córnea/irrigación sanguínea , Córnea/metabolismo , Córnea/patología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Endoteliales de la Vena Umbilical Humana/patología , Humanos , Integrina alfa5/genética , Integrina alfa5/metabolismo , Integrina beta1/genética , Integrina beta1/metabolismo , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/metabolismo , Masculino , Melanoma Experimental/metabolismo , Melanoma Experimental/patología , Ratones , Ratones Noqueados , Metástasis de la Neoplasia , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/deficiencia , Proteína Quinasa C/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de SeñalRESUMEN
Rho family GTPases act as molecular switches to regulate a range of physiological functions, including the regulation of the actin-based cytoskeleton, membrane trafficking, cell morphology, nuclear gene expression, and cell growth. Rho function is regulated by its ability to bind GTP and by its localization. We previously demonstrated functional and physical interactions between Rho3 and the clathrin-associated adaptor protein-1 (AP-1) complex, which revealed a role of Rho3 in regulating Golgi/endosomal trafficking in fission yeast. Sip1, a conserved AP-1 accessory protein, recruits the AP-1 complex to the Golgi/endosomes through physical interaction. In this study, we showed that Sip1 is required for Rho3 localization. First, overexpression of rho3⺠suppressed defective membrane trafficking associated with sip1-i4 mutant cells, including defects in vacuolar fusion, Golgi/endosomal trafficking and secretion. Notably, Sip1 interacted with Rho3, and GFP-Rho3, similar to Apm1-GFP, did not properly localize to the Golgi/endosomes in sip1-i4 mutant cells at 27°C. Interestingly, the C-terminal region of Sip1 is required for its localization to the Golgi/endosomes, because Sip1-i4-GFP protein failed to properly localize to Golgi/endosomes, whereas the fluorescence of Sip1ΔN mutant protein co-localized with that of FM4-64. Consistently, in the sip1-i4 mutant cells, which lack the C-terminal region of Sip1, binding between Apm1 and Rho3 was greatly impaired, presumably due to mislocalization of these proteins in the sip1-i4 mutant cells. Furthermore, the interaction between Apm1 and Rho3 as well as Rho3 localization to the Golgi/endosomes were significantly rescued in sip1-i4 mutant cells by the expression of Sip1ΔN. Taken together, these results suggest that Sip1 recruits Rho3 to the Golgi/endosomes through physical interaction and enhances the formation of the Golgi/endosome AP-1/Rho3 complex, thereby promoting crosstalk between AP-1 and Rho3 in the regulation of Golgi/endosomal trafficking in fission yeast.