Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.11 (AMPK)
 12,425 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A 57-kDa protein (p57) has been purified to homogeneity from a microsomal fraction of rat spleen. It is specifically and efficiently phosphorylated by the Src-like tyrosine kinase Lyn purified from the same source with a Km of 0.34 microM. The tyrosine kinases c-Fgr, Fyn, C-terminal Src kinase and p72syk, as well as the Ser/Thr-specific cAMP-dependent protein kinase and protein kinases CK1 and CK2 do not phosphorylate p57. C-terminal Src kinase, which acts to down-regulate the Src-like protein-tyrosine kinases, almost completely prevents the protein phosphorylation catalysed by Lyn. Protein mass fingerprinting with tryptic fragments identified p57 as a protein related to protein disulfide-isomerase which belongs to the superfamily of Cys-Gly-His-Cys-containing sequences. Lyn phosphorylates tyrosine residues Y444, Y453 and Y466 which are located in a highly acidic region of the protein at the C-terminus. Upon phosphorylation, p57 forms a complex with Lyn which can be immunoprecipitated with anti-Lyn IgG. The association which occurs between the phosphorylated substrate and the SH2 domain of the kinase is consistent with the suggested 'processive phosphorylation' model, which implies that a primary phosphorylation site of the substrate binds to the SH2 domain of the enzyme and triggers the phosphorylation at secondary site(s).
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PMID:Isolation from spleen of a 57-kDa protein substrate of the tyrosine kinase Lyn. Identification as a protein related to protein disulfide-isomerase and localisation of the phosphorylation sites. 863 26

Phosphorylation sites for casein kinase I were introduced into chimeric monoclonal antibody CC49 (MAb-chCC49) by inserting a synthetic fragment (CK1) encoding two casein kinase I phosphorylation sites into an expression vector. The phosphorylation sites were created by incorporating the predicted consensus sequences for phosphorylation by the casein kinase I at the carboxyl terminus of the heavy-chain constant region of the MAb-chCC49. The resultant modified MAb-chCC49 (MAb-chCC49CK1) was expressed and purified. The MAb-chCC49CK1 protein can be phosphorylated by the casein kinase I with [gamma-32P]ATP to high radiospecific activity. The 32P-labeled MAb-chCC49CK1 protein binds to cells expressing TAG-72 antigens. The introduction of phosphorylation sites into MAb provides new reagents for the diagnosis and treatment of cancer. This demonstrates that, as was described for the cAMP-dependent protein kinase site, the casein kinase I recognition site can also be used to introduce phosphorylation sites into proteins.
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PMID:Construction of phosphorylatable chimeric monoclonal antibody CC49 with a casein kinase I recognition site. 1002 74

Trypanosoma evansi contains protein kinases capable of phosphorylating endogenous substrates with apparent molecular masses in the range between 20 and 205 kDa. The major phosphopolypeptide band, pp55, was predominantly localized in the particulate fraction. Anti-alpha and anti-beta tubulin monoclonal antibodies recognized pp55 by Western blot analyses, suggesting that this band corresponds to phosphorylated tubulin. Inhibition experiments in the presence of emodin, heparin, and 2,3-bisphosphoglycerate indicated that the parasite tubulin kinase was a casein kinase 2 (CK2)-like activity. GTP, which can be utilized instead of ATP by CK2, stimulated rather than inactivated the phosphorylation of tubulin in the parasite homogenate and particulate fraction. However, GTP inhibited the cytosolic CK2 responsible for phosphorylating soluble tubulin and other soluble substrates. Casein and two selective peptide substrates, P1 (RRKDLHDDEEDEAMSITA) for casein kinase (CK1) and P2 (RRRADDSDDDDD) for CK2, were recognized as substrates in T. evansi. While the enzymes present in the soluble fraction predominantly phosphorylated P1, P2 was preferentially labeled in the particulate fractions. These results demonstrated the existence of CK1-like and CK2-like activities primarily located in the parasite cytosolic and membranous fractions, respectively. Histone II-A and kemptide (LRRASVA) also behaved as suitable substrates, implying the existence of other Ser/Thr kinases in T. evansi. Cyclic AMP only increased the phosphorylation of histone II-A and kemptide in the cytosol, demonstrating the existence of soluble cAMP-dependent protein kinase-like activities in T. evansi. However, no endogenous substrates for this enzyme were identified in this fraction. Further evidences were obtained by using PKI (6-22), a reported inhibitor of the catalytic subunit of mammalian cAMP-dependent protein kinases, which specifically hindered the cAMP-dependent phosphorylation of histone II-A and kemptide in the parasite soluble fraction. Since the sum of the values obtained in the parasite cytosolic and particulate fractions were always higher than the values observed in the total T. evansi lysate, the kinase activities examined here appeared to be inhibited in the original extract.
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PMID:Identification of casein kinase 1, casein kinase 2, and cAMP-dependent protein kinase-like activities in Trypanosoma evansi. 1576 1

The involvement of CK1 (casein kinase 1) delta in the regulation of multiple cellular processes implies a tight regulation of its activity on many different levels. At the protein level, reversible phosphorylation plays an important role in modulating the activity of CK1delta. In the present study, we show that PKA (cAMP-dependent protein kinase), Akt (protein kinase B), CLK2 (CDC-like kinase 2) and PKC (protein kinase C) alpha all phosphorylate CK1delta. PKA was identified as the major cellular CK1deltaCK (CK1delta C-terminal-targeted protein kinase) for the phosphorylation of CK1delta in vitro and in vivo. This was implied by the following evidence: PKA was detectable in the CK1deltaCK peak fraction of fractionated MiaPaCa-2 cell extracts, PKA shared nearly identical kinetic properties with those of CK1deltaCK, and both PKA and CK1deltaCK phosphorylated CK1delta at Ser370 in vitro. Furthermore, phosphorylation of CK1delta by PKA decreased substrate phosphorylation of CK1delta in vitro. Mutation of Ser370 to alanine increased the phosphorylation affinity of CK1delta for beta-casein and the GST (gluthatione S-transferase)-p53 1-64 fusion protein in vitro and enhanced the formation of an ectopic dorsal axis during Xenopus laevis development. Anchoring of PKA and CK1delta to centrosomes was mediated by AKAP (A-kinase-anchoring protein) 450. Interestingly, pre-incubation of MiaPaCa-2 cells with the synthetic peptide St-Ht31, which prevents binding between AKAP450 and the regulatory subunit RII of PKA, resulted in a 6-fold increase in the activity of CK1delta. In summary, we conclude that PKA phosphorylates CK1delta, predominantly at Ser370 in vitro and in vivo, and that site-specific phosphorylation of CK1delta by PKA plays an important role in modulating CK1delta-dependent processes.
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PMID:Phosphorylation of CK1delta: identification of Ser370 as the major phosphorylation site targeted by PKA in vitro and in vivo. 3100 Jun 25

Resistance to cisplatin (CDDP) is a major cause of cancer treatment failure, including human breast cancer. The tumor suppressor protein p53 is a key factor in the induction of cell cycle arrest, DNA repair, and apoptosis in response to cellular stimuli. This protein is phosphorylated in serine 15 and serine 20 during DNA damage repair or in serine 46 to induce apoptosis. Resveratrol (Resv) is a natural compound representing a promising chemosensitizer for cancer treatment that has been shown to sensitize tumor cells through upregulation and phosphorylation of p53 and inhibition of RAD51. We developed a CDDP-resistant MCF-7 cell line variant (MCF-7R) to investigate the effect of Resv in vitro in combination with CDDP over the role of p53 in overcoming CDDP resistance in MCF-7R cells. We have shown that Resv induces sensitivity to CDDP in MCF-7 and MCF-7R cells and that the downregulation of p53 protein expression and inhibition of p53 protein activity enhances resistance to CDDP in both cell lines. On the other hand, we found that Resv induces serine 20 (S20) phosphorylation in chemoresistant cells to activate p53 target genes such as PUMA and BAX, restoring apoptosis. It also changed the ratio between BCL-2 and BAX, where BCL-2 protein expression was decreased and at the same time BAX protein was increased. Interestingly, Resv attenuates CDDP-induced p53 phosphorylation in serine 15 (S15) and serine 46 (S46) probably through dephosphorylation and deactivation of ATM. It also activates different kinases, such as CK1, CHK2, and AMPK to induce phosphorylation of p53 in S20, suggesting a novel mechanism of p53 activation and chemosensitization to CDDP.
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PMID:Induction of p53 Phosphorylation at Serine 20 by Resveratrol Is Required to Activate p53 Target Genes, Restoring Apoptosis in MCF-7 Cells Resistant to Cisplatin. 3014 17

Although helminth parasites cause enormous suffering worldwide we know little of how protein phosphorylation, one of the most important post-translational modifications used for molecular signalling, regulates their homeostasis and function. This is particularly the case for schistosomes. Herein, we report a deep phosphoproteome exploration of adult Schistosoma mansoni, providing one of the richest phosphoprotein resources for any parasite so far, and employ the data to build the first parasite-specific kinomic array. Complementary phosphopeptide enrichment strategies were used to detect 15,844 unique phosphopeptides mapping to 3,176 proteins. The phosphoproteins were predicted to be involved in a wide range of biological processes and phosphoprotein interactome analysis revealed 55 highly interconnected clusters including those enriched with ribosome, proteasome, phagosome, spliceosome, glycolysis, and signalling proteins. 93 distinct phosphorylation motifs were identified, with 67 providing a 'footprint' of protein kinase activity; CaMKII, PKA and CK1/2 were highly represented supporting their central importance to schistosome function. Within the kinome, 808 phosphorylation sites were matched to 136 protein kinases, and 68 sites within 37 activation loops were discovered. Analysis of putative protein kinase-phosphoprotein interactions revealed canonical networks but also novel interactions between signalling partners. Kinomic array analysis of male and female adult worm extracts revealed high phosphorylation of transformation:transcription domain associated protein by both sexes, and CDK and AMPK peptides by females. Moreover, eight peptides including protein phosphatase 2C gamma, Akt, Rho2 GTPase, SmTK4, and the insulin receptor were more highly phosphorylated by female extracts, highlighting their possible importance to female worm function. We envision that these findings, tools and methodology will help drive new research into the functional biology of schistosomes and other helminth parasites, and support efforts to develop new therapeutics for their control.
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PMID:Deep phosphoproteome analysis of Schistosoma mansoni leads development of a kinomic array that highlights sex-biased differences in adult worm protein phosphorylation. 3220 12