Differential Phosphorylation of the Transcription Factor WRKY33 by the Protein Kinases CPK5/CPK6 and MPK3/MPK6 Cooperatively Regulates Camalexin Biosynthesis in Arabidopsis

The addition of thrombopoietin (TPO) to HEL cells, cultured in a chemically defined serum-free medium, induced a rapid and dose-dependent phosphorylation of the transcription factor CREB on serine133 (PSer133), as detected by Western blot analysis. TPO also significantly increased the transactivation of CRE-dependent promoter, as determined in transient transfection experiments. On the other hand, neither erythropoietin (Epo; 1 to 10 U) nor hemin (10−7 mol/L) were able to significantly stimulate CREB-PSer133 or to activate CRE-promoter in HEL cells. Although pharmacological inhibitors of protein kinase C (chelerytrine and BIM) and protein kinase A (H-89) failed to block the TPO-mediated CREB phosphorylation, a specific inhibitor of the mitogen-activated protein kinases (PD98059) completely blocked the ability of TPO to stimulate CREB-PSer133. Moreover, PD98059 significantly decreased the ability of TPO to upregulate the surface expression of the αIIbβ3 megakaryocytic marker in HEL cells. In parallel, primary CD34+ hematopoietic cells were seeded in liquid cultures supplemented with 100 ng/mL of TPO and examined by immunofluorescence for the coexpression of αIIbβ3 and CREB-PSer133 at various time points. High levels of nuclear CREB-PSer133 were unequivocally demonstrated in αIIbβ3+cells, including morphologically recognizable megakaryocytes. Taken together, these data suggest that CREB plays a role in modulating the expression of genes critical for megakaryocyte differentiation and that the TPO-mediated CREB phosphorylation seems to be regulated via mitogen-activated protein kinases.

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Gene- and cell-type-specific effects of signal transduction cascades on metal-regulated gene transcription appear to be independent of changes in the phosphorylation of metal-response-element-binding transcription factor-1

Biochemical Journal ◽

10.1042/bj20040504 ◽

2004 ◽

Vol 382 (1) ◽

pp. 33-41 ◽

Cited By ~ 25

Author(s):

Huimin JIANG ◽

Kai FU ◽

Glen K. ANDREWS

Keyword(s):

Transcription Factor ◽

Protein Kinase C ◽

Protein Kinase ◽

Protein Kinases ◽

Cell Type ◽

Specific Effects ◽

Kinase C ◽

Cell Type Specific ◽

Transcription Factor 1

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.

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Differential phosphorylation of ribosomal acidic proteins from yeast cell by two endogenous protein kinases: casein kinase-2 and 60S kinase.

Acta Biochimica Polonica ◽

10.18388/abp.1995_4634 ◽

1995 ◽

Vol 42 (3) ◽

pp. 357-362 ◽

Cited By ~ 10

Author(s):

R Szyszka ◽

A Boguszewska ◽

N Grankowski ◽

J P Ballesta

Keyword(s):

Protein Kinases ◽

Ribosomal Subunit ◽

Casein Kinase ◽

Casein Kinase 2 ◽

Amino Acid Sequences ◽

The Other ◽

Endogenous Protein ◽

Target Amino Acid ◽

Acidic Proteins ◽

Differential Phosphorylation

The native 80S ribosomes isolated from Saccharomyces cerevisiae (strain W303) cells was phosphorylated by two endogenous protein kinases: multifunctional casein kinase-2 (CK-2) and specific 60S kinase. Three acidic proteins within the 60S ribosomal subunit: YP1 beta, YP1 beta' and YP2 alpha are phosphorylated by both kinases. The other two proteins: YP1 alpha and YP2 beta are predominantly phosphorylated by CK-2 but not by 60S kinase. This was confirmed in the experiment with the recombinant protein, YP2 beta, as a substrate, which is practically not phosphorylated by specific 60S kinase. These results together with the previous data based on the target amino-acid sequences suggest that, in addition to the multifunctional casein kinase-2 and specific 60S kinase, there exist probably other protein kinase(s) which phosphorylate the ribosomal acidic proteins in the cell.

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