The protein kinase inhibitor SB203580 uncouples PMA-induced differentiation of HL-60 cells from phosphorylation of Hsp27

Abstract

HL-60 cells are an attractive model for studies of human myeloid cell differentiation. Among the well-examined parameters correlated to differentiation of HL-60 cells are the expression and phosphorylation of the small heat shock protein Hsp27. Here we demonstrate that PMA treatment of HL-60 cells stimulates different MAP kinase cascades, leading to significant activation of ERK2 and p38 reactivating kinase (p38RK). Using the protein kinase inhibitor SB 203580, we specifically inhibited p38RK and, thereby, activation of its target MAP kinase-activated protein kinase 2 (MAPKAP kinase 2), which is the major enzyme responsible for small Hsp phosphorylation. As a result, PMA-induced Hsp27 phosphorylation is inhibited in SB 203580-treated HL-60 cells indicating that p38RK and MAPKAP kinase 2 are components of the PMA-induced signal transduction pathway leading to Hsp27 phosphorylation. We further demonstrate that, although PMA-induced phosphorylation is inhibited, SB 203580-treated HL-60 cells are still able to differentiate to the macrophage-like phenotype as judged by decrease in cell proliferation, induction of expression of the cell surface antigen CD11b and changes in cell morphology. These results indicate that, although correlated, Hsp27 phosphorylation is not required for HL-60 cell differentiation. However, the results do not exclude that increased Hsp27 expression is involved in HL-60 cell differentiation.

Heat shock protein 27 phosphorylation: kinases, phosphatases, functions and pathology

“…However, the p38 MAPK inhibitor SB203580 could prevent PMA-induced Hsp27 phosphorylation, but did not abrogate differentiation, making it unlikely that Hsp27 phosphorylation is absolutely required for PMA-induced HL-60 cell differentiation [145]. Other studies have reported altered Hsp27 protein levels in differentiated cells [23,24,47,102], but few have addressed the correlation with changed Hsp27 phosphorylation [10,136].…”

“…Because PMA-induced differentiation of HL-60 cells was correlated with increased expression and phosphorylation of Hsp27, it was speculated that phosphorylation of Hsp27 is required for differentiation [145]. However, the p38 MAPK inhibitor SB203580 could prevent PMA-induced Hsp27 phosphorylation, but did not abrogate differentiation, making it unlikely that Hsp27 phosphorylation is absolutely required for PMA-induced HL-60 cell differentiation [145].…”

The hsp27kD heat shock protein and p38-MAPK signaling are required for regular epidermal differentiation

“…Stress related phosphorylation and dephorphorylation of hsp27 is catalyzed by enzymes of the p38-MAPK pathway [13,14]. Since specific pharmacological inhibitors of this signaling pathway are available they have been used to study the phosphorylation processes of hsp27 [15,16]. Unphosphorylated hsp27 forms large multimers, whereas its phosphorylation results in complex dissociation, altered protein binding [17,18] and loss of chaperoning activity [19,20].…”

“…Unphosphorylated hsp27 forms large multimers, whereas its phosphorylation results in complex dissociation, altered protein binding [17,18] and loss of chaperoning activity [19,20]. Hsp27 can be phosphorylated reversibly on three serine residues (15,78,82) by the mitogenactivated protein kinase-activated kinases 2 and 3 (MK2/3), which are activated through the p38 signaling pathway [21].…”

Introducing Professor Nikki Holbrook, Stress Signaling and Aging Section Editor

“…Furthermore, geldanamycin, an inhibitor of Hsp90, blocks kinase-mediated signaling events during T-lymphocyte activation (Schnaider et al 2000). Gaestel and coworkers (Schultz et al 1997) provided evidence that p38RK and MAPKAP kinase 2 are part of a signal transduction pathway leading to Hsp27 phosphorylation in HL-60 cells but that this phosphorylation event is not needed for HL-60 cell differentiation. A number of papers have added to our understanding of induced cytoprotection (thermotolerance), links between the stress response and apoptotic pathways, and blocking of signal transduction pathways.…”