Regulation of Neuroprotective Action of Vasoactive Intestinal Peptide in the Murine Developing Brain by Protein Kinase C and Mitogen-Activated Protein Kinase Cascades: In Vivo and In Vitro Studies

14-3-3 proteins may function as adapter or scaffold proteins in signal transduction pathways. We reported previously that several 14-3-3 isotypes bind to protein kinase C (PKC)-ζ and facilitate coupling of PKC-ζ to Raf-1 [van der Hoeven, van der Wal, Ruurs, van Dijk and van Blitterswijk (2000) Biochem. J. 345, 297-306], an event that boosts the mitogen-activated protein kinase (ERK) pathway in Rat-1 fibroblasts. The present work investigated whether bound 14-3-3 would affect PKC-ζ activity. Using recombinant 14-3-3 proteins and purified PKC-ζ in a convenient, newly developed in vitro kinase assay, we found that 14-3-3 proteins stimulated PKC-ζ activity in a dose-dependent fashion up to approx. 2.5-fold. Activation of PKC-ζ by 14-3-3 isotypes was unrelated to their mutual affinity, estimated by co-immunoprecipitation from COS cell lysates. Accordingly, PKC-ζ with a defective (point-mutated) 14-3-3-binding site, showed the same 14-3-3-stimulated activity as wild-type PKC-ζ. As 14-13-3 proteins are acidic, we tested several other acidic proteins, which turned out to stimulate PKC-ζ activity in a similar fashion, whereas neutral or basic proteins did not. These effects were not restricted to the atypical PKC-ζ, but were also found for classical PKC. Together, the results suggest that the stimulation of PKC activity by 14-3-3 proteins is non-specific and solely due to the acidic nature of these proteins, quite similar to that known for acidic lipids.

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Protein Kinase C and Cerebral Vasospasm

Journal of Cerebral Blood Flow & Metabolism ◽

10.1097/00004647-200108000-00001 ◽

2001 ◽

Vol 21 (8) ◽

pp. 887-906 ◽

Cited By ~ 108

Author(s):

Ismail Laher ◽

John H. Zhang

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Cerebral Vasospasm ◽

Protein Tyrosine Kinase ◽

Vascular Reactivity ◽

Receptor Activation ◽

Mitogen Activated Protein Kinase ◽

Kinase C ◽

Mitogen Activated Protein

Twenty-five years after the discovery of protein kinase C (PKC), the physiologic function of PKC, and especially its role in pathologic conditions, remains a subject of great interest with 30,000 studies published on these aspects. In the cerebral circulation, PKC plays a role in the regulation of myogenic tone by sensitization of myofilaments to calcium. Protein kinase C phosphorylates various ion channels including augmenting voltage-dependent Ca2+ channels and inhibiting K+ channels, which both lead to vessel contraction. These actions of PKC amplify vascular reactivity to different agonists and may be critical in the regulation of cerebral artery tone during vasospasm. Evidence accumulated during at least the last decade suggest that activation of PKC in cerebral vasospasm results in a delayed but prolonged contraction of major arteries after subarachnoid hemorrhage. Most of the experimental results in vitro or in animal models support the view that PKC is involved in cerebral vasospasm. Implication of PKC in cerebral vasospasm helps explain increased arterial narrowing at the signal transduction level and alters current perceptions that the pathophysiology is caused by a combination of multiple receptor activation, hemoglobin toxicity, and damaged neurogenic control. Activation of protein kinase C also interacts with other signaling pathways such as myosin light chain kinase, nitric oxide, intracellular Ca2+, protein tyrosine kinase, and its substrates such as mitogen-activated protein kinase. Even though identifying PKC revolutionized the understanding of cerebral vasospasm, clinical advances are hampered by the lack of clinical trials using selective PKC inhibitors.

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