AKT1 mediates multiple phosphorylation events that functionally promote HSF1 activation

Area of Science:

• Molecular Biology
• Cellular Stress Response

Background:

• The heat stress response involves the transcription factor heat shock factor 1 (HSF1), which upregulates heat shock proteins to maintain proteome integrity.
• HSF1 activation is a complex process requiring nuclear localization, trimerization, DNA binding, phosphorylation, and gene transactivation.

Purpose of the Study:

• To investigate the role of specific kinases, particularly AKT1, in regulating HSF1 activity through phosphorylation.
• To identify novel phosphorylation sites on HSF1 and elucidate their functional significance in transcriptional regulation.

Main Methods:

• Kinase assays to determine the ability of AKT1, AKT2, mTOR, p38, MEK1, and DYRK2 to phosphorylate HSF1 at S326.
• Mass spectrometry to identify all phosphorylation sites on HSF1 following AKT1 treatment.
• Functional assays to assess the impact of specific phosphorylation sites on HSF1 trimerization, gene transactivation, and TFIIB/CDK9 recruitment.

Main Results:

• AKT1 was identified as the most potent activator of HSF1 transcriptional activity through S326 phosphorylation, independent of mTOR.
• Mass spectrometry revealed that AKT1 phosphorylates HSF1 at T142, S230, S326, and T527.
• Phosphorylation at T142 is essential for HSF1 trimerization, while S230, S326, and T527 are critical for gene transactivation and the recruitment of TFIIB and CDK9.

Conclusions:

• HSF1 hyperphosphorylation is a targeted process, with specific residues playing direct roles in regulating its transcriptional activity.
• The novel phosphorylation site T527 in the transactivation domain highlights a new regulatory mechanism for HSF1.
• AKT1 plays a central role in HSF1 activation through multi-site phosphorylation, impacting key steps in the heat stress response.