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Def-6, a novel regulator of small GTPases in podocytes, acts downstream of atypical protein kinase C (aPKC) ?/?.

ABSTRACT: The atypical protein kinase C (aPKC) isotypes PKC?/? and PKC? are both expressed in podocytes; however, little is known about differences in their function. Previous studies in mice have demonstrated that podocyte-specific loss of PKC?/? leads to a severe glomerular phenotype, whereas mice deficient in PKC? develop no renal phenotype. We analyzed various effects caused by PKC?/? and PKC? deficiency in cultured murine podocytes. In contrast to PKC?-deficient podocytes, PKC?/?-deficient podocytes exhibited a severe actin cytoskeletal phenotype, reduced cell size, decreased number of focal adhesions, and increased activation of small GTPases. Comparative microarray analysis revealed that the guanine nucleotide exchange factor Def-6 was specifically up-regulated in PKC?/?-deficient podocytes. In vivo Def-6 expression is significantly increased in podocytes of PKC?/?-deficient mice. Cultured PKC?/?-deficient podocytes exhibited an enhanced membrane association of Def-6, indicating enhanced activation. Overexpression of aPKC?/? in PKC?/?-deficient podocytes could reduce the membrane-associated expression of Def-6 and rescue the actin phenotype. In the present study, PKC?/? was identified as an important factor for actin cytoskeletal regulation in podocytes and Def-6 as a specific downstream target of PKC?/? that regulates the activity of small GTPases and subsequently the actin cytoskeleton of podocytes.

SUBMITTER: Worthmann K

PROVIDER: S-EPMC5707189 | biostudies-literature | 2013 Dec

REPOSITORIES: biostudies-literature

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Def-6, a novel regulator of small GTPases in podocytes, acts downstream of atypical protein kinase C (aPKC) λ/ι.

Worthmann Kirstin K Leitges Michael M Teng Beina B Sestu Marcello M Tossidou Irini I Samson Thomas T Haller Hermann H Huber Tobias B TB Schiffer Mario M

The American journal of pathology 20131003 6

The atypical protein kinase C (aPKC) isotypes PKCλ/ι and PKCζ are both expressed in podocytes; however, little is known about differences in their function. Previous studies in mice have demonstrated that podocyte-specific loss of PKCλ/ι leads to a severe glomerular phenotype, whereas mice deficient in PKCζ develop no renal phenotype. We analyzed various effects caused by PKCλ/ι and PKCζ deficiency in cultured murine podocytes. In contrast to PKCζ-deficient podocytes, PKCλ/ι-deficient podocytes ...[more]

PMID: 24096077

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Project description:Defining the mechanisms of Mycobacterium tuberculosis (Mtb) persistence in the host macrophage and identifying mycobacterial factors responsible for it are keys to better understand tuberculosis pathogenesis. The emerging picture from ongoing studies of macrophage deactivation by Mtb suggests that ingested bacilli secrete various virulence determinants that alter phagosome biogenesis, leading to arrest of Mtb vacuole interaction with late endosomes and lysosomes. While most studies focused on Mtb interference with various regulators of the endosomal compartment, little attention was paid to mechanisms by which Mtb neutralizes early macrophage responses such as the NADPH oxidase (NOX2) dependent oxidative burst. Here we applied an antisense strategy to knock down Mtb nucleoside diphosphate kinase (Ndk) and obtained a stable mutant (Mtb Ndk-AS) that displayed attenuated intracellular survival along with reduced persistence in the lungs of infected mice. At the molecular level, pull-down experiments showed that Ndk binds to and inactivates the small GTPase Rac1 in the macrophage. This resulted in the exclusion of the Rac1 binding partner p67(phox) from phagosomes containing Mtb or Ndk-coated latex beads. Exclusion of p67(phox) was associated with a defect of both NOX2 assembly and production of reactive oxygen species (ROS) in response to wild type Mtb. In contrast, Mtb Ndk-AS, which lost the capacity to disrupt Rac1-p67(phox) interaction, induced a strong ROS production. Given the established link between NOX2 activation and apoptosis, the proportion of Annexin V positive cells and levels of intracellular active caspase 3 were significantly higher in cells infected with Mtb Ndk-AS compared to wild type Mtb. Thus, knock down of Ndk converted Mtb into a pro-apoptotic mutant strain that has a phenotype of increased susceptibility to intracellular killing and reduced virulence in vivo. Taken together, our in vitro and in vivo data revealed that Ndk contributes significantly to Mtb virulence via attenuation of NADPH oxidase-mediated host innate immunity.

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Def-6, a novel regulator of small GTPases in podocytes, acts downstream of atypical protein kinase C (aPKC) ?/?.

Publications

Def-6, a novel regulator of small GTPases in podocytes, acts downstream of atypical protein kinase C (aPKC) λ/ι.

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