scholarly journals Evidence That Atypical Protein Kinase C-λ and Atypical Protein Kinase C-ζ Participate in Ras-mediated Reorganization of the F-actin Cytoskeleton

1999 ◽  
Vol 144 (3) ◽  
pp. 413-425 ◽  
Author(s):  
Florian Überall ◽  
Karina Hellbert ◽  
Sonja Kampfer ◽  
Karl Maly ◽  
Andreas Villunger ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Actin Cytoskeleton ◽  
Dominant Negative ◽  
Stress Fibers ◽  
Atypical Protein Kinase C ◽  
Nih3t3 Cells ◽  
Atypical Protein Kinase ◽  
Kinase C ◽  
Actin Stress Fibers

Expression of transforming Ha-Ras L61 in NIH3T3 cells causes profound morphological alterations which include a disassembly of actin stress fibers. The Ras-induced dissolution of actin stress fibers is blocked by the specific PKC inhibitor GF109203X at concentrations which inhibit the activity of the atypical aPKC isotypes λ and ζ, whereas lower concentrations of the inhibitor which block conventional and novel PKC isotypes are ineffective. Coexpression of transforming Ha-Ras L61 with kinase-defective, dominant-negative (DN) mutants of aPKC-λ and aPKC-ζ, as well as antisense constructs encoding RNA-directed against isotype-specific 5′ sequences of the corresponding mRNA, abrogates the Ha-Ras–induced reorganization of the actin cytoskeleton. Expression of a kinase-defective, DN mutant of cPKC-α was unable to counteract Ras with regard to the dissolution of actin stress fibers. Transfection of cells with constructs encoding constitutively active (CA) mutants of atypical aPKC-λ and aPKC-ζ lead to a disassembly of stress fibers independent of oncogenic Ha-Ras. Coexpression of (DN) Rac-1 N17 and addition of the phosphatidylinositol 3′-kinase (PI3K) inhibitors wortmannin and LY294002 are in agreement with a tentative model suggesting that, in the signaling pathway from Ha-Ras to the cytoskeleton aPKC-λ acts upstream of PI3K and Rac-1, whereas aPKC-ζ functions downstream of PI3K and Rac-1. This model is supported by studies demonstrating that cotransfection with plasmids encoding L61Ras and either aPKC-λ or aPKC-ζ results in a stimulation of the kinase activity of both enzymes. Furthermore, the Ras-mediated activation of PKC-ζ was abrogated by coexpression of DN Rac-1 N17.

scholarly journals Mapping of Atypical Protein Kinase C within the Nerve Growth Factor Signaling Cascade: Relationship to Differentiation and Survival of PC12 Cells

2000 ◽  
Vol 20 (13) ◽  
pp. 4494-4504 ◽  
Author(s):  
Marie W. Wooten ◽  
M. Lamar Seibenhener ◽  
Kimberly B. W. Neidigh ◽  
Michel L. Vandenplas
Keyword(s):  
Protein Kinase C ◽  
Nerve Growth Factor ◽  
Growth Factor ◽  
Protein Kinase ◽  
Pc12 Cells ◽  
Dominant Negative ◽  
Atypical Protein Kinase C ◽  
Nerve Growth ◽  
Atypical Protein Kinase ◽  
Kinase C

ABSTRACT The pathway by which atypical protein kinase C (aPKC) contributes to nerve growth factor (NGF) signaling is poorly understood. We previously reported that in PC12 cells NGF-induced activation of mitogen-activated protein kinase (MAPK) occurs independently of classical and nonclassical PKC isoforms, whereas aPKC isoforms were shown to be required for NGF-induced differentiation. NGF-induced activation of PKC-ι was observed to be dependent on phosphatidylinositol 3-kinase (PI3K) and led to coassociation of PKC-ι with Ras and Src. Expression of dominant negative mutants of either Src (DN2) or Ras (Asn-17) impaired activation of PKC-ι by NGF. At the level of Raf-1, neither PKC-ι nor PI3 kinase was required for activation; however, PKC-ι could weakly activate MEK. Inhibitors of PKC-ι activity and PI3K had no effect on NGF-induced MAPK or p38 activation but reduced NGF-stimulated c-Jun N-terminal kinase activity. Src, PI3K, and PKC-ι were likewise required for NGF-induced NF-κB activation and cell survival, whereas Ras was not required for either survival or NF-κB activation but was required for differentiation. IKK existed as a complex with PKC-ι, Src and IκB. Consistent with a role for Src in regulating NF-κB activation, an absence of Src activity impaired recruitment of PKC-ι into an IKK complex and markedly impaired NGF-induced translocation of p65/NF-κB to the nucleus. These findings reveal that in PC12 cells, aPKCs comprise a molecular switch to regulate differentiation and survival responses coupled downstream to NF-κB. On the basis of these findings, Src emerges as a critical upstream regulator of both PKC-ι and the NF-κB pathway.

scholarly journals Role of Atypical Protein Kinase C in Estradiol-Triggered G1/S Progression of MCF-7 Cells

2004 ◽  
Vol 24 (17) ◽  
pp. 7643-7653 ◽  
Author(s):  
Gabriella Castoria ◽  
Antimo Migliaccio ◽  
Marina Di Domenico ◽  
Maria Lombardi ◽  
Antonietta de Falco ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Nuclear Translocation ◽  
Dominant Negative ◽  
Serine Phosphorylation ◽  
Atypical Protein Kinase C ◽  
Atypical Protein Kinase ◽  
Cos Cells ◽  
Kinase C ◽  
Mcf 7

ABSTRACT Expression of a dominant negative atypical protein kinase C (aPKC), PKCζ, prevents nuclear translocation of extracellular regulated kinase 2 (ERK-2), p27 nuclear reduction, and DNA synthesis induced by estradiol in human mammary cancer-derived MCF-7 cells. aPKC action upstream of these events has been analyzed. In hormone-stimulated NIH 3T3 and Cos cells ectopically expressing human estrogen receptor alpha (hERα), aPKC is activated by phosphatidylinositol 3-kinase (PI 3-kinase) and, in turn, controls the Ras/MEK-1/ERK cascade. In MCF-7 and Cos cells stimulated by hormone, PI 3-kinase activates PKCζ by Thr410 phosphorylation. Serine phosphorylation of PKCζ is simultaneously induced. PKCζ activation leads to recruitment of Ras to a multimolecular complex that also includes hERα, Src, PI 3-kinase, and aPKC. We propose that PKCζ pushes Ras and the signaling complex close together in such a way that it facilitates the Src-dependent Ras activation. This activation is crucial for the interplay between estradiol-triggered signaling and cell cycle machinery.

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