Dataset Information

Proplatelet generation in the mouse requires PKC?-dependent RhoA inhibition.

ABSTRACT: During thrombopoiesis, megakaroycytes undergo extensive cytoskeletal remodeling to form proplatelet extensions that eventually produce mature platelets. Proplatelet formation is a tightly orchestrated process that depends on dynamic regulation of both tubulin reorganization and Rho-associated, coiled-coil containing protein kinase/RhoA activity. A disruption in tubulin dynamics or RhoA activity impairs proplatelet formation and alters platelet morphology. We previously observed that protein kinase Cepsilon (PKC?), a member of the protein kinase C family of serine/threonine-kinases, expression varies during human megakaryocyte differentiation and modulates megakaryocyte maturation and platelet release. Here we used an in vitro model of murine platelet production to investigate a potential role for PKC? in proplatelet formation. By immunofluorescence we observed that PKC? colocalizes with ?/?-tubulin in specific areas of the marginal tubular-coil in proplatelets. Moreover, we found that PKC? expression escalates during megakarocyte differentiation and remains elevated in proplatelets, whereas the active form of RhoA is substantially downregulated in proplatelets. PKC? inhibition resulted in lower proplatelet numbers and larger diameter platelets in culture as well as persistent RhoA activation. Finally, we demonstrate that pharmacological inhibition of RhoA is capable of reversing the proplatelet defects mediated by PKC? inhibition. Collectively, these data indicate that by regulating RhoA activity, PKC? is a critical mediator of mouse proplatelet formation in vitro.

SUBMITTER: Gobbi G

PROVIDER: S-EPMC3952532 | biostudies-literature | 2013 Aug

REPOSITORIES: biostudies-literature

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Proplatelet generation in the mouse requires PKCε-dependent RhoA inhibition.

Gobbi Giuliana G Mirandola Prisco P Carubbi Cecilia C Masselli Elena E Sykes Stephen M SM Ferraro Francesca F Nouvenne Antonio A Thon Jonathan N JN Italiano Joseph E JE Vitale Marco M

Blood 20130709 7

During thrombopoiesis, megakaroycytes undergo extensive cytoskeletal remodeling to form proplatelet extensions that eventually produce mature platelets. Proplatelet formation is a tightly orchestrated process that depends on dynamic regulation of both tubulin reorganization and Rho-associated, coiled-coil containing protein kinase/RhoA activity. A disruption in tubulin dynamics or RhoA activity impairs proplatelet formation and alters platelet morphology. We previously observed that protein kina ...[more]

PMID: 23838351

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Project description:Adjusting to a wide range of light intensities is an essential feature of retinal rod bipolar cell (RBC) function. While persuasive evidence suggests this modulation involves phosphorylation by protein kinase C-alpha (PKC?), the targets of PKC? phosphorylation in the retina have not been identified. PKC? activity and phosphorylation in RBCs was examined by immunofluorescence confocal microscopy using a conformation-specific PKC? antibody and antibodies to phosphorylated PKC motifs. PKC? activity was dependent on light and expression of TRPM1, and RBC dendrites were the primary sites of light-dependent phosphorylation. PKC?-dependent retinal phosphoproteins were identified using a phosphoproteomics approach to compare total protein and phosphopeptide abundance between phorbol ester-treated wild type and PKC? knockout (PKC?-KO) mouse retinas. Phosphopeptide mass spectrometry identified over 1100 phosphopeptides in mouse retina, with 12 displaying significantly greater phosphorylation in WT compared to PKC?-KO samples. The differentially phosphorylated proteins fall into the following functional groups: cytoskeleton/trafficking (4 proteins), ECM/adhesion (2 proteins), signaling (2 proteins), transcriptional regulation (3 proteins), and homeostasis/metabolism (1 protein). Two strongly differentially expressed phosphoproteins, BORG4 and TPBG, were localized to the synaptic layers of the retina, and may play a role in PKC?-dependent modulation of RBC physiology. Data are available via ProteomeXchange with identifier PXD012906. SIGNIFICANCE: Retinal rod bipolar cells (RBCs), the second-order neurons of the mammalian rod visual pathway, are able to modulate their sensitivity to remain functional across a wide range of light intensities, from starlight to daylight. Evidence suggests that this modulation requires the serine/threonine kinase, PKC?, though the specific mechanism by which PKC? modulates RBC physiology is unknown. This study examined PKC? phosophorylation patterns in mouse rod bipolar cells and then used a phosphoproteomics approach to identify PKC?-dependent phosphoproteins in the mouse retina. A small number of retinal proteins showed significant PKC?-dependent phosphorylation, including BORG4 and TPBG, suggesting a potential contribution to PKC?-dependent modulation of RBC physiology.

Dataset Information

Proplatelet generation in the mouse requires PKC?-dependent RhoA inhibition.

Publications

Proplatelet generation in the mouse requires PKCε-dependent RhoA inhibition.

Similar Datasets

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