Limits...
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.

Show MeSH

Related in: MedlinePlus

32P-labeled proteins of the Detroit pharyngeal cell M/P fraction. Autoradiographs showing (A) 32P-labeled proteins of the M/P fraction after phosphorylation of intact pharyngeal cells, and (B) 32P-labeled  proteins after phosphorylation of the isolated M/P fraction, each then  treated with group A streptococci (gr A streptococci), or purified SDH. After  treatment, phosphorylation was carried out in the presence of [γ-32P]ATP.  Genistein (G), staurosporine (S), or both (GS) were included in the reaction mixtures to determine the amino acid site specificity of the phosphorylation modifications. Phosphorylation of pharyngeal cells without any  prior treatment served as the control. Controls in which bacteria alone  were processed under similar conditions released no phosphorylated proteins in the soluble fraction (not shown). Each lane received ∼40 μg of  total protein. Figures on the left side are the molecular mass (kDa) of standard prestained proteins (GIBCO BRL). Major phosphorylated proteins  are indicated by molecular mass (kDa).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2199133&req=5

Figure 1: 32P-labeled proteins of the Detroit pharyngeal cell M/P fraction. Autoradiographs showing (A) 32P-labeled proteins of the M/P fraction after phosphorylation of intact pharyngeal cells, and (B) 32P-labeled proteins after phosphorylation of the isolated M/P fraction, each then treated with group A streptococci (gr A streptococci), or purified SDH. After treatment, phosphorylation was carried out in the presence of [γ-32P]ATP. Genistein (G), staurosporine (S), or both (GS) were included in the reaction mixtures to determine the amino acid site specificity of the phosphorylation modifications. Phosphorylation of pharyngeal cells without any prior treatment served as the control. Controls in which bacteria alone were processed under similar conditions released no phosphorylated proteins in the soluble fraction (not shown). Each lane received ∼40 μg of total protein. Figures on the left side are the molecular mass (kDa) of standard prestained proteins (GIBCO BRL). Major phosphorylated proteins are indicated by molecular mass (kDa).

Mentions: Because of its surface location, SDH may be predicted to directly contact the pharyngeal cell membrane or other cellular components, depending on the stage of pharyngeal infection by streptococci. Hence, we first compared the effect of whole streptococci or purified SDH on the phosphorylation of proteins in intact Detroit cells, in the presence of [γ-32P]ATP. After phosphorylation, the cells were lysed, and the resulting cell extract was then separated into cytosolic and M/P fractions, the latter containing the plasma membrane, organelles, and nucleosomes. In the absence or presence of whole streptococci/SDH, several proteins in M/P fraction were phosphorylated at varying intensities (Fig. 1 A), while the incorporation of 32P in cytosolic proteins was found to be very weak (data not shown). In the presence of intact streptococci or SDH, phosphorylation of several proteins in the M/P fraction was enhanced, with greater incorporation of 32P in the presence of streptococci than SDH (Fig. 1 A). Interestingly, phosphorylation of a 17-kD protein in this M/P fraction occurred only after its interaction with whole streptococci or SDH. While SDH-mediated phosphorylation of the 17-kD protein was completely inhibited by the tyrosine kinase inhibitor, genistein, and significantly inhibited by the mixture of 100 μM genistein and 1 μM staurosporine, it was partially inhibited in the case of whole streptococci–mediated phosphorylation. Staurosporine alone also reduced the streptococcal/SDH-mediated phosphorylation of the 180-kD, 100-kD, and completely inhibited that of the 30-kD and 22–24-kD pharyngeal cell proteins. These results indicate that pharyngeal cell phosphorylation mediated by whole streptococci is likely induced by SDH via the activation of both protein tyrosine kinase and other kinases, including protein kinase C.


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)

32P-labeled proteins of the Detroit pharyngeal cell M/P fraction. Autoradiographs showing (A) 32P-labeled proteins of the M/P fraction after phosphorylation of intact pharyngeal cells, and (B) 32P-labeled  proteins after phosphorylation of the isolated M/P fraction, each then  treated with group A streptococci (gr A streptococci), or purified SDH. After  treatment, phosphorylation was carried out in the presence of [γ-32P]ATP.  Genistein (G), staurosporine (S), or both (GS) were included in the reaction mixtures to determine the amino acid site specificity of the phosphorylation modifications. Phosphorylation of pharyngeal cells without any  prior treatment served as the control. Controls in which bacteria alone  were processed under similar conditions released no phosphorylated proteins in the soluble fraction (not shown). Each lane received ∼40 μg of  total protein. Figures on the left side are the molecular mass (kDa) of standard prestained proteins (GIBCO BRL). Major phosphorylated proteins  are indicated by molecular mass (kDa).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: 32P-labeled proteins of the Detroit pharyngeal cell M/P fraction. Autoradiographs showing (A) 32P-labeled proteins of the M/P fraction after phosphorylation of intact pharyngeal cells, and (B) 32P-labeled proteins after phosphorylation of the isolated M/P fraction, each then treated with group A streptococci (gr A streptococci), or purified SDH. After treatment, phosphorylation was carried out in the presence of [γ-32P]ATP. Genistein (G), staurosporine (S), or both (GS) were included in the reaction mixtures to determine the amino acid site specificity of the phosphorylation modifications. Phosphorylation of pharyngeal cells without any prior treatment served as the control. Controls in which bacteria alone were processed under similar conditions released no phosphorylated proteins in the soluble fraction (not shown). Each lane received ∼40 μg of total protein. Figures on the left side are the molecular mass (kDa) of standard prestained proteins (GIBCO BRL). Major phosphorylated proteins are indicated by molecular mass (kDa).
Mentions: Because of its surface location, SDH may be predicted to directly contact the pharyngeal cell membrane or other cellular components, depending on the stage of pharyngeal infection by streptococci. Hence, we first compared the effect of whole streptococci or purified SDH on the phosphorylation of proteins in intact Detroit cells, in the presence of [γ-32P]ATP. After phosphorylation, the cells were lysed, and the resulting cell extract was then separated into cytosolic and M/P fractions, the latter containing the plasma membrane, organelles, and nucleosomes. In the absence or presence of whole streptococci/SDH, several proteins in M/P fraction were phosphorylated at varying intensities (Fig. 1 A), while the incorporation of 32P in cytosolic proteins was found to be very weak (data not shown). In the presence of intact streptococci or SDH, phosphorylation of several proteins in the M/P fraction was enhanced, with greater incorporation of 32P in the presence of streptococci than SDH (Fig. 1 A). Interestingly, phosphorylation of a 17-kD protein in this M/P fraction occurred only after its interaction with whole streptococci or SDH. While SDH-mediated phosphorylation of the 17-kD protein was completely inhibited by the tyrosine kinase inhibitor, genistein, and significantly inhibited by the mixture of 100 μM genistein and 1 μM staurosporine, it was partially inhibited in the case of whole streptococci–mediated phosphorylation. Staurosporine alone also reduced the streptococcal/SDH-mediated phosphorylation of the 180-kD, 100-kD, and completely inhibited that of the 30-kD and 22–24-kD pharyngeal cell proteins. These results indicate that pharyngeal cell phosphorylation mediated by whole streptococci is likely induced by SDH via the activation of both protein tyrosine kinase and other kinases, including protein kinase C.

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