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Regulation of the phosphorylation of human pharyngeal cell proteins by group A streptococcal surface dehydrogenase: signal transduction between streptococci and pharyngeal cells.

Pancholi V, Fischetti VA - J. Exp. Med. (1997)

Bottom Line: Intact streptococci and purified SDH induce a similar protein phosphorylation pattern with the de novo tyrosine phosphorylation of a 17-kD protein found in the membrane/particulate fraction of the pharyngeal cells.Treatment of pharyngeal cells with protein kinase inhibitors such as genistein and staurosporine significantly inhibited streptococcal invasion of pharyngeal cells.To identify the membrane receptor that elicits these signaling events, we found that SDH bound specifically to 30- and 32-kD membrane proteins in a direct ligand-binding assay.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Bacterial Pathogenesis and Immunology, The Rockefeller University, New York 10021, USA. panchov@rockvax.rockefeller.edu

ABSTRACT
Whether cell-to-cell communication results when group A streptococci interact with their target cells is unknown. Here, we report that upon contact with cultured human pharyngeal cells, both whole streptococci and purified streptococcal surface dehydrogenase (SDH) activate pharyngeal cell protein tyrosine kinase as well as protein kinase C, thus regulating the phosphorylation of cellular proteins. SDH, a major surface protein of group A streptococci, has both glyceraldehyde-3-phosphate dehydrogenase and ADP-ribosylating enzyme activities that may relate to early stages of streptococcal infection. Intact streptococci and purified SDH induce a similar protein phosphorylation pattern with the de novo tyrosine phosphorylation of a 17-kD protein found in the membrane/particulate fraction of the pharyngeal cells. However, this phosphorylation required the presence of cytosolic components. NH2-terminal amino acid sequence analysis identified the 17-kD protein as nuclear core histone H3. Both phosphotyrosine and phosphoserine-specific monoclonal antibodies reacted with the 17-kD protein by Western blot, suggesting that the binding of SDH to these pharyngeal cells elicits a novel signaling pathway that ultimately leads to activation of histone H3-specific kinases. Genistein-inhibitable phosphorylation of histone H3 indicates that tyrosine kinase plays a key role in this event. Treatment of pharyngeal cells with protein kinase inhibitors such as genistein and staurosporine significantly inhibited streptococcal invasion of pharyngeal cells. Therefore, these data indicated that streptococci/SDH-mediated phosphorylation plays a critical role in bacterial entry into the host cell. To identify the membrane receptor that elicits these signaling events, we found that SDH bound specifically to 30- and 32-kD membrane proteins in a direct ligand-binding assay. These findings clearly suggest that SDH plays an important role in cellular communication between streptococci and pharyngeal cells that may be important in host cell gene transcription, and hence in the pathogenesis of streptococcal infection.

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Identification of the 17-kD phosphorylated protein. (A) Coomassie stain and autoradiograph of a PVDF membrane showing the immunoprecipitated phosphorylated 17-kD protein with antiphosphotyrosine monoclonal antibodies. Detroit cells were treated with SDH, and the phosphorylation reaction was carried out as described in Fig. 1. Pharyngeal cell M/P fraction and cytosolic proteins were then immunoprecipitated with antiphosphotyrosine monoclonal antibodies. Proteins in the precipitate were resolved by 11% SDS-PAGE and electrotransferred to a PVDF membrane. The arrows  indicate the phosphorylated 17-kD membrane protein closely migrating with three other coprecipitated nonphosphorylated membrane proteins. (B) Direct immunostaining of the immunoprecipitated 17-kD protein complex on Western blot. The blots were stained with with antiphosphotyrosine and antiphosphoserine mouse monoclonal antibodies followed by HRP-labeled antimouse IgG conjugate and detected with ECL Western blotting detection  reagents. The phosphorylated 17-kD protein as detected by this method is marked (arrow). (C) NH2-terminal amino acid sequence of the immunoprecipitated 17-kD phosphorylated protein and its comparison with the NH2-terminal sequence of human histone H3 and other representative eukaryotic histone H3 proteins.
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Figure 3: Identification of the 17-kD phosphorylated protein. (A) Coomassie stain and autoradiograph of a PVDF membrane showing the immunoprecipitated phosphorylated 17-kD protein with antiphosphotyrosine monoclonal antibodies. Detroit cells were treated with SDH, and the phosphorylation reaction was carried out as described in Fig. 1. Pharyngeal cell M/P fraction and cytosolic proteins were then immunoprecipitated with antiphosphotyrosine monoclonal antibodies. Proteins in the precipitate were resolved by 11% SDS-PAGE and electrotransferred to a PVDF membrane. The arrows indicate the phosphorylated 17-kD membrane protein closely migrating with three other coprecipitated nonphosphorylated membrane proteins. (B) Direct immunostaining of the immunoprecipitated 17-kD protein complex on Western blot. The blots were stained with with antiphosphotyrosine and antiphosphoserine mouse monoclonal antibodies followed by HRP-labeled antimouse IgG conjugate and detected with ECL Western blotting detection reagents. The phosphorylated 17-kD protein as detected by this method is marked (arrow). (C) NH2-terminal amino acid sequence of the immunoprecipitated 17-kD phosphorylated protein and its comparison with the NH2-terminal sequence of human histone H3 and other representative eukaryotic histone H3 proteins.

Mentions: To isolate SDH-mediated tyrosine phosphorylated pharyngeal cell proteins, intact pharyngeal cells were treated with SDH (as in Fig. 1), and [32P]ATP-labeled M/P proteins were immunoprecipitated using antiphosphotyrosine monoclonal antibodies after cell lysis and fractionation. We found that the labeled 17-kD protein was immunoprecipitated along with three other closely migrating nonlabeled proteins (Fig. 3 A). To further confirm that the immunoprecipitated 17-kD protein is tyrosine phosphorylated, Western blots containing the labeled 17-kD proteins were reacted with either antiphosphotyrosine or antiphosphoserine antibodies. We found that both monoclonal antibodies bound to the 17-kD protein, with stronger reactivity exhibited by the antiphosphotyrosine antibody (Fig. 3 A). These results, while confirming the tyrosine phosphorylation of the 17-kD protein, also revealed that this protein is phosphorylated at serine residues (Fig. 3 B).


Regulation of the phosphorylation of human pharyngeal cell proteins by group A streptococcal surface dehydrogenase: signal transduction between streptococci and pharyngeal cells.

Pancholi V, Fischetti VA - J. Exp. Med. (1997)

Identification of the 17-kD phosphorylated protein. (A) Coomassie stain and autoradiograph of a PVDF membrane showing the immunoprecipitated phosphorylated 17-kD protein with antiphosphotyrosine monoclonal antibodies. Detroit cells were treated with SDH, and the phosphorylation reaction was carried out as described in Fig. 1. Pharyngeal cell M/P fraction and cytosolic proteins were then immunoprecipitated with antiphosphotyrosine monoclonal antibodies. Proteins in the precipitate were resolved by 11% SDS-PAGE and electrotransferred to a PVDF membrane. The arrows  indicate the phosphorylated 17-kD membrane protein closely migrating with three other coprecipitated nonphosphorylated membrane proteins. (B) Direct immunostaining of the immunoprecipitated 17-kD protein complex on Western blot. The blots were stained with with antiphosphotyrosine and antiphosphoserine mouse monoclonal antibodies followed by HRP-labeled antimouse IgG conjugate and detected with ECL Western blotting detection  reagents. The phosphorylated 17-kD protein as detected by this method is marked (arrow). (C) NH2-terminal amino acid sequence of the immunoprecipitated 17-kD phosphorylated protein and its comparison with the NH2-terminal sequence of human histone H3 and other representative eukaryotic histone H3 proteins.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199133&req=5

Figure 3: Identification of the 17-kD phosphorylated protein. (A) Coomassie stain and autoradiograph of a PVDF membrane showing the immunoprecipitated phosphorylated 17-kD protein with antiphosphotyrosine monoclonal antibodies. Detroit cells were treated with SDH, and the phosphorylation reaction was carried out as described in Fig. 1. Pharyngeal cell M/P fraction and cytosolic proteins were then immunoprecipitated with antiphosphotyrosine monoclonal antibodies. Proteins in the precipitate were resolved by 11% SDS-PAGE and electrotransferred to a PVDF membrane. The arrows indicate the phosphorylated 17-kD membrane protein closely migrating with three other coprecipitated nonphosphorylated membrane proteins. (B) Direct immunostaining of the immunoprecipitated 17-kD protein complex on Western blot. The blots were stained with with antiphosphotyrosine and antiphosphoserine mouse monoclonal antibodies followed by HRP-labeled antimouse IgG conjugate and detected with ECL Western blotting detection reagents. The phosphorylated 17-kD protein as detected by this method is marked (arrow). (C) NH2-terminal amino acid sequence of the immunoprecipitated 17-kD phosphorylated protein and its comparison with the NH2-terminal sequence of human histone H3 and other representative eukaryotic histone H3 proteins.
Mentions: To isolate SDH-mediated tyrosine phosphorylated pharyngeal cell proteins, intact pharyngeal cells were treated with SDH (as in Fig. 1), and [32P]ATP-labeled M/P proteins were immunoprecipitated using antiphosphotyrosine monoclonal antibodies after cell lysis and fractionation. We found that the labeled 17-kD protein was immunoprecipitated along with three other closely migrating nonlabeled proteins (Fig. 3 A). To further confirm that the immunoprecipitated 17-kD protein is tyrosine phosphorylated, Western blots containing the labeled 17-kD proteins were reacted with either antiphosphotyrosine or antiphosphoserine antibodies. We found that both monoclonal antibodies bound to the 17-kD protein, with stronger reactivity exhibited by the antiphosphotyrosine antibody (Fig. 3 A). These results, while confirming the tyrosine phosphorylation of the 17-kD protein, also revealed that this protein is phosphorylated at serine residues (Fig. 3 B).

Bottom Line: Intact streptococci and purified SDH induce a similar protein phosphorylation pattern with the de novo tyrosine phosphorylation of a 17-kD protein found in the membrane/particulate fraction of the pharyngeal cells.Treatment of pharyngeal cells with protein kinase inhibitors such as genistein and staurosporine significantly inhibited streptococcal invasion of pharyngeal cells.To identify the membrane receptor that elicits these signaling events, we found that SDH bound specifically to 30- and 32-kD membrane proteins in a direct ligand-binding assay.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Bacterial Pathogenesis and Immunology, The Rockefeller University, New York 10021, USA. panchov@rockvax.rockefeller.edu

ABSTRACT
Whether cell-to-cell communication results when group A streptococci interact with their target cells is unknown. Here, we report that upon contact with cultured human pharyngeal cells, both whole streptococci and purified streptococcal surface dehydrogenase (SDH) activate pharyngeal cell protein tyrosine kinase as well as protein kinase C, thus regulating the phosphorylation of cellular proteins. SDH, a major surface protein of group A streptococci, has both glyceraldehyde-3-phosphate dehydrogenase and ADP-ribosylating enzyme activities that may relate to early stages of streptococcal infection. Intact streptococci and purified SDH induce a similar protein phosphorylation pattern with the de novo tyrosine phosphorylation of a 17-kD protein found in the membrane/particulate fraction of the pharyngeal cells. However, this phosphorylation required the presence of cytosolic components. NH2-terminal amino acid sequence analysis identified the 17-kD protein as nuclear core histone H3. Both phosphotyrosine and phosphoserine-specific monoclonal antibodies reacted with the 17-kD protein by Western blot, suggesting that the binding of SDH to these pharyngeal cells elicits a novel signaling pathway that ultimately leads to activation of histone H3-specific kinases. Genistein-inhibitable phosphorylation of histone H3 indicates that tyrosine kinase plays a key role in this event. Treatment of pharyngeal cells with protein kinase inhibitors such as genistein and staurosporine significantly inhibited streptococcal invasion of pharyngeal cells. Therefore, these data indicated that streptococci/SDH-mediated phosphorylation plays a critical role in bacterial entry into the host cell. To identify the membrane receptor that elicits these signaling events, we found that SDH bound specifically to 30- and 32-kD membrane proteins in a direct ligand-binding assay. These findings clearly suggest that SDH plays an important role in cellular communication between streptococci and pharyngeal cells that may be important in host cell gene transcription, and hence in the pathogenesis of streptococcal infection.

Show MeSH
Related in: MedlinePlus