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The HIV transactivator protein, Tat, enhances HIV transcription by recruiting P-TEFb from the inactive 7SK snRNP complex and directing it to proviral elongation complexes. To test the hypothesis that T-cell receptor (TCR) signaling induces critical post-translational modifications leading to enhanced interactions between P-TEFb and Tat, we employed affinity purification–tandem mass spectrometry to analyze P-TEFb. TCR or phorbal ester (PMA) signaling strongly induced phosphorylation of the CDK9 kinase at Ser175. Molecular modeling studies based on the Tat/P-TEFb X-ray structure suggested that pSer175 strengthens the intermolecular interactions between CDK9 and Tat. Mutations in Ser175 confirm that this residue could mediate critical interactions with Tat and with the bromodomain protein BRD4. The S175A mutation reduced CDK9 interactions with Tat by an average of 1.7-fold, but also completely blocked CDK9 association with BRD4. The phosphomimetic S175D mutation modestly enhanced Tat association with CDK9 while causing a 2-fold disruption in BRD4 association with CDK9. Since BRD4 is unable to compete for binding to CDK9 carrying S175A, expression of CDK9 carrying the S175A mutation in latently infected cells resulted in a robust Tat-dependent reactivation of the provirus. Similarly, the stable knockdown of BRD4 led to a strong enhancement of proviral expression. Immunoprecipitation experiments show that CDK9 phosphorylated at Ser175 is excluded from the 7SK RNP complex. Immunofluorescence and flow cytometry studies carried out using a phospho-Ser175-specific antibody demonstrated that Ser175 phosphorylation occurs during TCR activation of primary resting memory CD4+ T cells together with upregulation of the Cyclin T1 regulatory subunit of P-TEFb, and Thr186 phosphorylation of CDK9. We conclude that the phosphorylation of CDK9 at Ser175 plays a critical role in altering the competitive binding of Tat and BRD4 to P-TEFb and provides an informative molecular marker for the identification of the transcriptionally active form of P-TEFb., Author Summary The release of the transcription elongation factor P-TEFb from the 7SK RNP complex and its binding to the HIV Tat transactivator protein enables the efficient transcription of HIV proviruses. In resting memory T-cells, which carry the bulk of the latent HIV viral pool, limiting the cellular levels of P-TEFb ensures that the provirus remains silenced unless the host cell is activated. Here we demonstrate that T-cell receptor (TCR) activation induces phosphorylation of Ser175, a residue which is located at the interface between CycT1, CDK9 and Tat. Phosphorylation of Ser175 occurs on free or 7SK-dissociated P-TEFb and genetic experiments indicate that this modification enhances P-TEFb interaction with Tat resulting in Tat-dependent reactivation of HIV proviral transcription. Modification of Ser175 appears critical for controlling the competitive binding of Tat and the bromodomain protein BRD4 to P-TEFb. Activation of P-TEFb in resting T-cells thus involves both the initial assembly of the 7SK snRNP complex and the subsequent mobilization of P-TEFb by cellular signaling and Tat. Therefore, pSer175 provides an informative molecular marker for the identification of the transcriptionally active form of P-TEFb that can be used to monitor the extent of T-cell activation during therapeutic interventions aimed at virus eradication.

The 146-kDa Pasteurella multocida toxin (PMT) is the main virulence factor to induce P. multocida-associated progressive atrophic rhinitis in various animals. PMT leads to a destruction of nasal turbinate bones implicating an effect of the toxin on osteoblasts and/or osteoclasts. The toxin induces constitutive activation of Gα proteins of the Gq/11-, G12/13- and Gi-family by deamidating an essential glutamine residue. To study the PMT effect on bone cells, we used primary osteoblasts derived from rat calvariae and stromal ST-2 cells as differentiation model. As marker of functional osteoblasts the expression and activity of alkaline phosphatase, formation of mineralization nodules or expression of specific transcription factors as osterix was determined. Here, we show that the toxin inhibits differentiation and/or function of osteoblasts by activation of Gαq/11. Subsequently, Gαq/11 activates RhoA via p63RhoGEF, which specifically interacts with Gαq/11 but not with other G proteins like Gα12/13 and Gαi. Activated RhoA transactivates the mitogen-activated protein (MAP) kinase cascade via Rho kinase, involving Ras, MEK and ERK, resulting in inhibition of osteoblast differentiation. PMT-induced inhibition of differentiation was selective for the osteoblast lineage as adipocyte-like differentiation of ST-2 cells was not hampered. The present work provides novel insights, how the bacterial toxin PMT can control osteoblastic development by activating heterotrimeric G proteins of the Gαq/11-family and is a molecular pathogenetic basis for understanding the role of the toxin in bone loss during progressive atrophic rhinitis induced by Pasteurella multocida., Author Summary Pasteurella multocida causes as a facultative pathogen various diseases in men and animals. One induced syndrome is atrophic rhinitis, which is a form of osteopenia, mainly characterized by facial distortion due to degradation of nasal turbinate bones. Strains, which especially affect bone tissue, produce the protein toxin P. multocida toxin (PMT). Importantly, PMT alone is capable to induce all symptoms of atrophic rhinitis. To cause osteopenia PMT influences the development and/or activity of specialized bone cells like osteoblasts and osteoclasts. Recently, we could identify the molecular mechanism of PMT. The toxin constitutively activates certain heterotrimeric G proteins by deamidation. Here, we studied the effect of PMT on the differentiation of osteoblasts. We demonstrate the direct action of PMT on osteoblasts and osteoblast-like cells and as a consequence inhibition of osteoblastic differentiation. Moreover, we revealed the underlying signal transduction pathway to impair proper osteoblast development. We show that PMT activates small GTPases in a Gαq/11 dependent manner via a non-ubiquitously expressed RhoGEF. In turn the mitogen-activated protein kinase pathway is transactivated leading to inhibition of osteoblastogenesis. Our findings present a mechanism how PMT hijacks host cell signaling pathways to hinder osteoblast development, which contributes to the syndrome of atrophic rhinitis.