Dissecting stress-activated protein kinase (SAPK)-signaling pathways using multiplex gene knockout HeLa cells

Area of Science:

• Cellular Biology
• Molecular Biology
• Signal Transduction

Background:

• The stress-activated protein kinase (SAPK) pathway involves multiple JNK and p38 members with potentially overlapping functions.
• Dissecting the specific roles of individual SAPK isotypes is crucial for understanding complex cellular signaling networks.

Purpose of the Study:

• To elucidate the isotype-specific and overlapping functions of SAPK family members.
• To investigate the roles of JNK and p38 isotypes in regulating substrate phosphorylation and immediate-early gene expression.

Main Methods:

• CRISPR-Cas9 gene editing was used to generate HeLa-derived multiplex knockout (KO) cell lines, including triple, quadruple, septuple, and sole-survivor (ss) hextuple SAPK KO cells.
• Western blot analyses with phospho-specific antibodies were performed to assess substrate phosphorylation.
• Expression levels of immediate-early genes (c-Jun, EGR1, c-Fos) were analyzed under various stress conditions.

Main Results:

• p38 kinases were primarily responsible for phosphorylating MAPKAPK2, MSK1, Mnk1, and CREB/ATF-1, while JNK kinases mediated c-Jun/JunD phosphorylation.
• JNK1 was identified as the main contributor to c-Jun/JunD phosphorylation, and p38α was the predominant kinase for p38 substrates.
• JNK1/JNK2 controlled c-Jun expression, p38 predominantly regulated EGR1 and c-Fos expression, but cytokine-induced EGR1 expression showed complex regulation independent of JNK/p38.

Conclusions:

• The comprehensive multigene KO and ss-KO strategy effectively dissects complex intracellular signaling pathways with multiple family members.
• SAPK isotypes exhibit distinct and sometimes overlapping roles in regulating cellular responses to stress and stimuli.
• Further research is needed to fully understand the intricate mechanisms governing SAPK-mediated immediate-early gene expression.