Single Molecule Studies and Kinase Activity Measurements Reveal Regulatory Interactions between the Master Kinases Phosphoinositide-Dependent-Kinase-1 (PDK1), Protein Kinase B (AKT1/PKB) and Protein Kinase C (PKCα)

2021 ◽  
Author(s):  
Moshe T. Gordon ◽  
Brian P. Ziemba ◽  
Joseph J. Falke
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Single Molecule ◽  
Kinase Activity ◽  
Protein Kinase B ◽  
Regulatory Interactions ◽  
Kinase C ◽  
Activity Measurements ◽  
Single Molecule Studies ◽  
Phosphoinositide Dependent Kinase

scholarly journals Single-Molecule Studies Reveal a Hidden Key Step in the Activation Mechanism of Membrane-Bound Protein Kinase C-α

Biochemistry ◽  
2014 ◽  
Vol 53 (10) ◽  
pp. 1697-1713 ◽  
Author(s):  
Brian P. Ziemba ◽  
Jianing Li ◽  
Kyle E. Landgraf ◽  
Jefferson D. Knight ◽  
Gregory A. Voth ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Single Molecule ◽  
Activation Mechanism ◽  
Kinase C ◽  
Single Molecule Studies ◽  
Membrane Bound

scholarly journals Identification of Key Phospholipids That Bind and Activate Atypical PKCs

Biomedicines ◽  
2021 ◽  
Vol 9 (1) ◽  
pp. 45
Author(s):  
Suresh Velnati ◽  
Sara Centonze ◽  
Federico Girivetto ◽  
Daniela Capello ◽  
Ricardo M. Biondi ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phosphatidic Acid ◽  
Kinase Activity ◽  
Membrane Lipids ◽  
Atypical Protein Kinase C ◽  
Atypical Protein Kinase ◽  
Kinase C ◽  
Specific Regulation ◽  
Phosphatidylinositol 4

PKCζ and PKCι/λ form the atypical protein kinase C subgroup, characterised by a lack of regulation by calcium and the neutral lipid diacylglycerol. To better understand the regulation of these kinases, we systematically explored their interactions with various purified phospholipids using the lipid overlay assays, followed by kinase activity assays to evaluate the lipid effects on their enzymatic activity. We observed that both PKCζ and PKCι interact with phosphatidic acid and phosphatidylserine. Conversely, PKCι is unique in binding also to phosphatidylinositol-monophosphates (e.g., phosphatidylinositol 3-phosphate, 4-phosphate, and 5-phosphate). Moreover, we observed that phosphatidylinositol 4-phosphate specifically activates PKCι, while both isoforms are responsive to phosphatidic acid and phosphatidylserine. Overall, our results suggest that atypical Protein kinase C (PKC) localisation and activity are regulated by membrane lipids distinct from those involved in conventional PKCs and unveil a specific regulation of PKCι by phosphatidylinositol-monophosphates.

scholarly journals Inhibition of growth-factor-induced phosphorylation and activation of protein kinase B/Akt by atypical protein kinase C in breast cancer cells

2000 ◽  
Vol 352 (2) ◽  
pp. 475-482 ◽  
Author(s):  
Muling MAO ◽  
Xianjun FANG ◽  
Yiling LU ◽  
Ruth LAPUSHIN ◽  
Robert C. BAST ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Protein Kinase C ◽  
Growth Factor ◽  
Protein Kinase ◽  
Cancer Cells ◽  
Breast Cancer Cells ◽  
Protein Kinase B ◽  
Akt Phosphorylation ◽  
Kinase C ◽  
Pkc Inhibitors

The protein kinase B/Akt serine/threonine kinase, located downstream of phosphoinositide 3-kinase (PI-3K), is a major regulator of cellular survival and proliferation. Atypical protein kinase C (aPKC) family members are activated by PI-3K and also contribute to cell proliferation, suggesting that Akt and aPKC might interact to activate signalling through the PI-3K cascade. Here we demonstrate that blocking PKC activity in MDA-MB-468 breast cancer cells increased the phosphorylation and activity of Akt. Functional PI-3K was required for the PKC inhibitors to increase Akt phosphorylation and activation, potentially owing to the activation of specific PKC isoforms by PI-3K. The concentration dependence of the action of the PKC inhibitors implicates aPKC in the inhibition of Akt phosphorylation and activity. In support of a role for aPKC in the regulation of Akt, Akt and PKCζ or PKCλ/ℓ were readily co-precipitated from the BT-549 breast cancer cell line. Furthermore, the overexpression of PKCζ inhibited growth-factor-induced increases in Akt phosphorylation and activity. Thus PKCζ associates physically with Akt and decreases Akt phosphorylation and enzyme activity. The effects of PKC on Akt were transmitted through the PI-3K cascade as indicated by changes in p70 s6 kinase (p70s6k) phosphorylation. Thus PKCζ, and potentially other PKC isoenzymes, regulate growth-factor-mediated Akt phosphorylation and activation, which is consistent with a generalized role for PKCζ in limiting growth factor signalling through the PI-3K/Akt pathway.

scholarly journals Abnormal protein kinase C down regulation and reduced substrate levels in non-phorbol ester-responsive 3T3-TNR9 cells.

1990 ◽  
Vol 10 (5) ◽  
pp. 2122-2132 ◽  
Author(s):  
H P Biemann ◽  
R L Erikson
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phorbol Ester ◽  
Kinase Activity ◽  
Subcellular Location ◽  
Parental Cell ◽  
Down Regulation ◽  
Kinase C ◽  
Cell Variant ◽  
Swiss 3T3 Cell

The cell line TNR9 (E. Butler-Gralla and H. R. Herschman, J. Cell. Physiol. 107:59-67, 1981) in a Swiss 3T3 cell variant that expresses protein kinase C (PKC) but is mitogenically nonresponsive to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). We have found that PKCs purified from variant and parental cells are identical as judged by kinase activity, protease mapping, and column chromatography. We analyzed cellular levels and subcellular location of PKC in TPA-treated 3T3 and TNR9 cells via immunoprecipitation of [35S]methionine-labeled protein and assay of immune-complex PKC kinase activity. TNR9 cells grew to higher densities than parental 3T3 cells. TNR9 cells at maximal density did not down regulate PKC in response to long-term TPA treatment. We compared the 80-kilodalton (kDa) PKC substrate phosphorylation in 3T3 and TNR9 cells by using two-dimensional gels and found that TNR9 cells treated with TPA for 30 min contained only 10 to 15% as much 32Pi associated with the 80-kDa as did parental cells. The TNR9 80-kDa substrate was present at reduced levels compared with the parental-cell 80-kDa substrate as judged by immunoblot and silver staining. Thus, the loss of mitogenic responsiveness to TPA in TNR9 cells is accompanied by resistance to TPA-mediated down regulation of PKC and reduced phosphosubstrate levels.

CK1δ kinase activity is modulated by protein kinase C α (PKCα)-mediated site-specific phosphorylation

Amino Acids ◽  
2016 ◽  
Vol 48 (5) ◽  
pp. 1185-1197 ◽  
Author(s):  
Zhigang Meng ◽  
Joachim Bischof ◽  
Chiara Ianes ◽  
Doris Henne-Bruns ◽  
Pengfei Xu ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Kinase Activity ◽  
Site Specific ◽  
Kinase C

scholarly journals Activation of hepatocyte protein kinase C by redox-cycling quinones

1989 ◽  
Vol 260 (2) ◽  
pp. 499-507 ◽  
Author(s):  
G E Kass ◽  
S K Duddy ◽  
S Orrenius
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Kinase Activity ◽  
Specific Activity ◽  
Active Oxygen Species ◽  
Redox Cycling ◽  
Active Oxygen ◽  
Oxygen Species ◽  
Cytosolic Protein ◽  
Kinase C

The effects of quinone-generated active oxygen species on rat hepatocyte protein kinase C were investigated. The specific activity of cytosolic protein kinase C was increased 2-3-fold in hepatocytes incubated with the redox-cycling quinones, menadione, duroquinone or 2,3-dimethoxy-1,4-naphthoquinone, without alterations in particulate protein kinase C specific activity or Ca2+- and lipid-independent kinase activities. Redox-cycling quinones did not stimulate translocation of protein kinase C; however, activated protein kinase C was redistributed from cytosol to the particulate fraction when quinone-treated hepatocytes were exposed to 12-O-tetradecanoylphorbol 13-acetate (TPA). Quinone treatment did not alter cytosolic phorbol 12,13-dibutyrate (PDBu) binding capacity, and the cytosol of both control and quinone-treated hepatocytes exhibited a Kd for PDBu binding of 2 nM. Quinone-mediated activation of cytosolic protein kinase C was reversed by incubation with 10 mM-beta-mercaptoethanol, dithiothreitol or GSH, at 4 degrees C for 24 h. Furthermore, protein kinase C specific activity in control cytosol incubated in air increased by over 100% within 3 h; this increase was reversed by thiol-reducing agents. Similarly, incubation of partially-purified rat brain protein kinase C in air, or with low concentrations of GSSG in the presence of GSH, resulted in a 2-2.5-fold increase in Ca2+- and lipid-dependent kinase activity. In contrast with the effects of the redox-cycling quinones, when hepatocytes were treated with the thiol agents N-ethylmaleimide (NEM), p-benzoquinone (pBQ) or p-chloromercuribenzoic acid (pCMB), the cytosolic Ca2+- and lipid-dependent kinase activity was significantly inhibited, but the particulate-associated protein kinase C activity was unaffected. The Ca2+- and lipid-independent kinase activity of both the cytosolic and particulate fractions was significantly stimulated by NEM, but was unaffected by pBQ and pCMB. These results show that hepatocyte cytosolic protein kinase C is activated to a high-Vmax form by quinone-generated active oxygen species, and this effect is due to a reduction-sensitive modification of the thiol/disulphide status of protein kinase C.

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