scholarly journals A Chemical-genetics and Nanoparticle Enabled Approach for in vivo Protein Kinase Analysis

2020 ◽  
Author(s):  
Fengqian Chen ◽  
Qi Liu ◽  
Terrell Hilliard ◽  
Tingzeng Wang ◽  
Hongjun Liang ◽  
...  

AbstractThe human kinome contains >500 protein kinases, and regulates up to 30% of the proteome. Kinase study is currently hindered by a lack of in vivo analysis approaches due to two factors: our inability to distinguish the kinase reaction of interest from those of other kinases in live cells and the cell impermeability of the ATP analogs. Herein, we tackled this issue by combining the widely used chemical genetic method developed by Dr. Kevan Shokat and colleagues with nanoparticle-mediated intracellular delivery of the ATP analog. The critical AKT1 protein kinase, which has been successfully studied with the method, was used as our initial prototype. Briefly, enlargement of the ATP binding pocket, by mutating the gate-keeper Methionine residue to a Glycine, prompted the mutant AKT1 to preferentially use the bulky ATP analog N6-Benzyl-ATP-γ-S (A*TPγS) and, thus, differentiating AKT1-catalyzed and other phosphorylation events. The lipid/calcium/phosphate (LCP) nanoparticle was used for efficient intracellular delivery of A*TPγS, overcoming the cell impermeability issue. The mutant, but not wild-type, AKT1 used the delivered A*TPγS for autophosphorylation and phosphorylating its substrates in live cells. Thus, an in vivo protein kinase analysis method has been developed. The strategy should be widely applicable to other protein kinases.

Molecules ◽  
2021 ◽  
Vol 26 (15) ◽  
pp. 4599
Author(s):  
Augustine Ahmadu ◽  
Claire Delehouzé ◽  
Anas Haruna ◽  
Lukman Mustapha ◽  
Bilqis Lawal ◽  
...  

The purpose of this work is to investigate the protein kinase inhibitory activity of constituents from Acacia auriculiformis stem bark. Column chromatography and NMR spectroscopy were used to purify and characterize betulin from an ethyl acetate soluble fraction of acacia bark. Betulin, a known inducer of apoptosis, was screened against a panel of 16 disease-related protein kinases. Betulin was shown to inhibit Abelson murine leukemia viral oncogene homolog 1 (ABL1) kinase, casein kinase 1ε (CK1ε), glycogen synthase kinase 3α/β (GSK-3 α/β), Janus kinase 3 (JAK3), NIMA Related Kinase 6 (NEK6), and vascular endothelial growth factor receptor 2 kinase (VEGFR2) with activities in the micromolar range for each. The effect of betulin on the cell viability of doxorubicin-resistant K562R chronic myelogenous leukemia cells was then verified to investigate its putative use as an anti-cancer compound. Betulin was shown to modulate the mitogen-activated protein (MAP) kinase pathway, with activity similar to that of imatinib mesylate, a known ABL1 kinase inhibitor. The interaction of betulin and ABL1 was studied by molecular docking, revealing an interaction of the inhibitor with the ABL1 ATP binding pocket. Together, these data demonstrate that betulin is a multi-target inhibitor of protein kinases, an activity that can contribute to the anticancer properties of the natural compound and to potential treatments for leukemia.


2006 ◽  
Vol 27 (1) ◽  
pp. 170-181 ◽  
Author(s):  
N. Ronkina ◽  
A. Kotlyarov ◽  
O. Dittrich-Breiholz ◽  
M. Kracht ◽  
E. Hitti ◽  
...  

ABSTRACT MK2 and MK3 represent protein kinases downstream of p38 mitogen-activated protein kinase (MAPK). Deletion of the MK2 gene in mice resulted in an impaired inflammatory response although MK3, which displays extensive structural similarities and identical functional properties in vitro, is still present. Here, we analyze tumor necrosis factor (TNF) production and expression of p38 MAPK and tristetraprolin (TTP) in MK3-deficient mice and demonstrate that there are no significant differences with wild-type animals. We show that in vivo MK2 and MK3 are expressed and activated in parallel. However, the level of activity of MK2 is always significantly higher than that of MK3. Accordingly, we hypothesized that MK3 could have significant effects only in an MK2-free background and generated MK2/MK3 double-knockout mice. Unexpectedly, these mice are viable and show no obvious defects due to loss of compensation between MK2 and MK3. However, there is a further reduction of TNF production and expression of p38 and TTP in double-knockout mice compared to MK2-deficient mice. This finding, together with the observation that ectopically expressed MK3 can rescue MK2 deficiency similarly to MK2, indicates that both kinases share the same physiological function in vivo but are expressed to different levels.


2005 ◽  
Vol 389 (1) ◽  
pp. 127-135 ◽  
Author(s):  
Claire E. EYERS ◽  
Helen McNEILL ◽  
Axel KNEBEL ◽  
Nick MORRICE ◽  
Simon J. C. ARTHUR ◽  
...  

A protein expressed in immune cells and muscle was detected in muscle extracts as a substrate for several SAPKs (stress-activated protein kinases). It interacted specifically with the F-actin capping protein CapZ in splenocytes, and was therefore termed ‘CapZIP’ (CapZ-interacting protein). Human CapZIP was phosphorylated at Ser-179 and Ser-244 by MAPKAP-K2 (mitogen-activated protein kinase-activated protein kinase 2) or MAPKAP-K3 in vitro. Anisomycin induced the phosphorylation of CapZIP at Ser-179 in Jurkat cells, which was prevented by SB 203580, consistent with phosphorylation by MAPKAP-K2 and/or MAPKAP-K3. However, osmotic shock-induced phosphorylation of Ser-179 was unaffected by SB 203580. These and other results suggest that CapZIP is phosphorylated at Ser-179 in cells by MAPKAP-K2/MAPKAP-K3, and at least one other protein kinase. Stress-activated MAP kinase family members phosphorylated human CapZIP at many sites, including Ser-68, Ser-83, Ser-108 and Ser-216. Ser-108 became phosphorylated when Jurkat cells were exposed to osmotic shock, which was unaffected by SB 203580 and/or PD 184352, or in splenocytes from mice that do not express either SAPK3/p38γ or SAPK4/p38δ. Our results suggest that CapZIP may be phosphorylated by JNK (c-Jun N-terminal kinase), which phosphorylates CapZIP to >5 mol/mol within minutes in vitro. Osmotic shock or anisomycin triggered the dissociation of CapZIP from CapZ in Jurkat cells, suggesting that phosphorylation of CapZIP may regulate the ability of CapZ to remodel actin filament assembly in vivo.


1999 ◽  
Vol 19 (2) ◽  
pp. 1569-1581 ◽  
Author(s):  
Cathy Tournier ◽  
Alan J. Whitmarsh ◽  
Julie Cavanagh ◽  
Tamera Barrett ◽  
Roger J. Davis

ABSTRACT The c-Jun NH2-terminal protein kinase (JNK) is a member of the mitogen-activated protein kinase (MAPK) group and is an essential component of a signaling cascade that is activated by exposure of cells to environmental stress. JNK activation is regulated by phosphorylation on both Thr and Tyr residues by a dual-specificity MAPK kinase (MAPKK). Two MAPKKs, MKK4 and MKK7, have been identified as JNK activators. Genetic studies demonstrate that MKK4 and MKK7 serve nonredundant functions as activators of JNK in vivo. We report here the molecular cloning of the gene that encodes MKK7 and demonstrate that six isoforms are created by alternative splicing to generate a group of protein kinases with three different NH2 termini (α, β, and γ isoforms) and two different COOH termini (1 and 2 isoforms). The MKK7α isoforms lack an NH2-terminal extension that is present in the other MKK7 isoforms. This NH2-terminal extension binds directly to the MKK7 substrate JNK. Comparison of the activities of the MKK7 isoforms demonstrates that the MKK7α isoforms exhibit lower activity, but a higher level of inducible fold activation, than the corresponding MKK7β and MKK7γ isoforms. Immunofluorescence analysis demonstrates that these MKK7 isoforms are detected in both cytoplasmic and nuclear compartments of cultured cells. The presence of MKK7 in the nucleus was not, however, required for JNK activation in vivo. These data establish that theMKK4 and MKK7 genes encode a group of protein kinases with different biochemical properties that mediate activation of JNK in response to extracellular stimuli.


2006 ◽  
Vol 401 (1) ◽  
pp. 29-38 ◽  
Author(s):  
Gopal P. Sapkota ◽  
Lorna Cummings ◽  
Felicity S. Newell ◽  
Christopher Armstrong ◽  
Jennifer Bain ◽  
...  

Hormones and growth factors induce the activation of a number of protein kinases that belong to the AGC subfamily, including isoforms of PKA, protein kinase B (also known as Akt), PKC, S6K p70 (ribosomal S6 kinase), RSK (p90 ribosomal S6 kinase) and MSK (mitogen- and stress-activated protein kinase), which then mediate many of the physiological processes that are regulated by these extracellular agonists. It can be difficult to assess the individual functions of each AGC kinase because their substrate specificities are similar. Here we describe the small molecule BI-D1870, which inhibits RSK1, RSK2, RSK3 and RSK4 in vitro with an IC50 of 10–30 nM, but does not signi-ficantly inhibit ten other AGC kinase members and over 40 other protein kinases tested at 100-fold higher concentrations. BI-D1870 is cell permeant and prevents the RSK-mediated phorbol ester- and EGF (epidermal growth factor)-induced phosphoryl-ation of glycogen synthase kinase-3β and LKB1 in human embry-onic kidney 293 cells and Rat-2 cells. In contrast, BI-D1870 does not affect the agonist-triggered phosphorylation of substrates for six other AGC kinases. Moreover, BI-D1870 does not suppress the phorbol ester- or EGF-induced phosphorylation of CREB (cAMP-response-element-binding protein), consistent with the genetic evidence indicating that MSK, and not RSK, isoforms mediate the mitogen-induced phosphorylation of this transcription factor.


2006 ◽  
Vol 394 (3) ◽  
Author(s):  
Mark H. Rider

The AMPK (AMP-activated protein kinase)-related protein kinase subfamily of the human kinome comprises 12 members closely related to the catalytic α1/α2 subunits of AMPK. The precise role of the AMPK-related kinases and their in vivo substrates is rather unclear at present, but some are involved in regulating cell polarity, whereas others appear to control cellular differentiation. Of the 12 human AMPK-related protein kinase family members, 11 can be activated following phosphorylation of their T-loop threonine residue by the LKB1 complex. Nine of these AMPK-related kinases activated by LKB1 contain an UBA (ubiquitin-associated) domain immediately C-terminal to the kinase catalytic domain. In this issue of the Biochemical Journal, Jaleel et al. show that the presence of an UBA domain in AMP-related kinases allows LKB1-induced phosphorylation and activation. The findings have implications for understanding the molecular mechanisms of activation of this fascinating family of protein kinases. Also, mutations in the UBA domains of the AMP-related kinase genes might be present in families with Peutz–Jehgers syndrome and in other cancer patients.


1983 ◽  
Vol 213 (2) ◽  
pp. 281-288 ◽  
Author(s):  
D F Qi ◽  
R C Schatzman ◽  
G J Mazzei ◽  
R S Turner ◽  
R L Raynor ◽  
...  

Effects of polyamines on various protein kinases were investigated. It was found that both phospholipid-sensitive Ca2+-dependent protein kinase and myosin light-chain kinase (a calmodulin-sensitive species of Ca2+-dependent protein kinase) were inhibited to different degrees by polyamines, with an approximate order of inhibitory potency of spermine = 1, 12-diaminododecane greater than spermidine = 1, 10-diaminodecane much greater than cadaverine = putrescine. Kinetic analysis revealed that spermine inhibited the phospholipid-sensitive enzyme non-competitively with respect to Ca2+ (Ki = 0.84 mM) and phosphatidylserine (Ki = 0.90 mM); it also inhibited myosin light-chain kinase non-competitively with respect to Ca2+ (Ki = 1.82 mM) and calmodulin (Ki = 2.73 mM). 1, 12-Diaminododecane, in comparison, inhibited the phospholipid-sensitive enzyme competitively with respect to Ca2+ (Ki = 0.45 mM) and phosphatidylserine (Ki = 0.50 mM); it also inhibited myosin light-chain kinase competitively with respect to calmodulin (Ki = 0.63 mM) but non-competitively with respect to Ca2+ (Ki = 1.49 mM). Moreover, spermine (0.5 mM) was found to inhibit markedly phosphatidylserine/Ca2+- and calmodulin/Ca2+-stimulated phosphorylation of endogenous proteins in rat brain particulate fraction. All the polyamines tested were practically without effect on cyclic AMP-dependent and cyclic GMP-dependent protein kinases. Polyarginine, like spermine, was found to be a more selective inhibitor of Ca2+-dependent protein kinases, whereas polyglutamate preferentially inhibited the cyclic nucleotide-dependent enzymes. The present results indicated that, in addition to certain lipophilic compounds (such as trifluoperazine, palmitoylcarnitine, adriamycin and naphthalenesulphonamide) and polypeptides with hydrophobic regions (such as melittin and polymyxin B) previously reported, polycationic compounds (exemplified by polyamines) could also inhibit the two classes of Ca2+-dependent protein kinases requiring either phospholipid or calmodulin as a cofactor. Because of the high cellular concentration (up to 10 mM) and the differential effects of polyamines, it is suggested that spermine, and to smaller extents spermidine and putrescine, may be involved in the regulation of certain Ca2+-dependent protein-phosphorylation systems in vivo.


2011 ◽  
Vol 39 (2) ◽  
pp. 472-476 ◽  
Author(s):  
Angus J.M. Cameron

Targeting the protein kinase ATP-binding pocket provides a significant opportunity for the treatment of disease. Recent studies have revealed a central activity-independent role for nucleotide pocket occupation in the allosteric behaviour of diverse kinases. Regulation of nucleotide pocket conformation with either nucleotides or ATP competitive inhibitors has revealed an added dimension to the targeting of kinases. In the present paper, using PKC (protein kinase C) as a paradigm, the liabilities and opportunities associated with the occupation of the nucleotide pocket are explored.


2004 ◽  
Vol 382 (1) ◽  
pp. 33-41 ◽  
Author(s):  
Huimin JIANG ◽  
Kai FU ◽  
Glen K. ANDREWS

Post-translational modification of MTF-1 (metal-response-element-binding transcription factor-1) was suggested to play a role in its metalloregulatory functions. In the present study, pulse labelling and two-dimensional electrophoresis–Western blotting were used to demonstrate that, although MTF-1 is highly modified in vivo, its phosphorylation level does not rapidly change in response to metals, nor does its overall modification pattern. Recombinant MTF-1 was found to serve as an in vitro substrate for casein kinase II, c-Jun N-terminal kinase and protein kinase C, but inhibition of these kinases in vivo did not significantly change the modification pattern of MTF-1. Northern blotting revealed that inhibitors of casein kinase II and c-Jun N-terminal kinase severely attenuate the metal-induced transcription of the native chromatin-packaged metallothionein-I and zinc transporter-1 genes, whereas protein kinase C inhibitors exerted gene- and cell-type-specific effects on the metal regulation and basal expression of these two genes. A chromatin immunoprecipitation assay was used to demonstrate that none of these inhibitors prevent the metal-dependent recruitment of MTF-1 to the MT-I promoter. In brief, results of the present study suggest that protein kinases may not alter the phosphorylation state of MTF-1 during the rapid-response phase to metals, nor do they regulate the metal-dependent formation of a stable MTF-1–chromatin complex. Instead, protein kinases may exert their interdependent effects on metal-induced gene expression by acting on cofactors that interact with MTF-1.


2001 ◽  
Vol 276 (15) ◽  
pp. 12369-12377 ◽  
Author(s):  
Heidi Kieschnick ◽  
Therese Wakefield ◽  
Carl Anthony Narducci ◽  
Con Beckers

The role of calcium-dependent protein kinases in the invasion ofToxoplasma gondiiinto its animal host cells was analyzed. KT5926, an inhibitor of calcium-dependent protein kinases in other systems, is known to block the motility ofToxoplasmatachyzoites and their attachment to host cells.In vivo, KT5926 blocks the phosphorylation of only three parasite proteins, and in parasite extracts only a single KT5926-sensitive protein kinase activity was detected. This activity was calcium-dependent but did not require calmodulin. In a search for calcium-dependent protein kinases inToxoplasma, two members of the class of calmodulin-like domain protein kinases (CDPKs) were detected. TgCDPK2 was only expressed at the mRNA level in tachyzoites, but no protein was detected. TgCDPK1 protein was expressed inToxoplasmatachyzoites and cofractionated precisely with the peak of KT5926-sensitive protein kinase activity. TgCDPK1 kinase activity was calcium-dependent but did not require calmodulin or phospholipids. TgCDPK1 was found to be inhibited effectively by KT5926 at concentrations that block parasite attachment to host cells.In vitro, TgCDPK1 phosphorylated three parasite proteins that migrated identical to the three KT5926-sensitive phosphoproteins detectedin vivo. Based on these observations, a central role is suggested for TgCDPK1 in regulatingToxoplasmamotility and host cell invasion.


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