Differences in phosphorylation of human and chicken stathmin by MAP kinase

Abstract

Stathmin/Op18 is a highly conserved 19 kDa cytosolic phosphoprotein. Human and chicken stathmin share 93% identity with only 11 amino acid substitutions. One of the substituted amino acids is serine 25, which is a glycine in chicken stathmin. In human stathmin, serine 25 is the main phosphorylation site for MAP kinase. In this study, we have compared the phosphorylation of human and chicken stathmin. The proteins were expressed in Sf9 cells using the baculovirus expression system and purified for in vitro phosphorylation assays. Phosphorylation with MAP kinase showed that chicken stathmin was phosphorylated 10 times less than human stathmin. To identify the phosphorylation sites we used liquid chromatography/mass spectrometry (LC/MS/MS). The only amino acid found phosphorylated was serine 38, which corresponds to the minor phosphorylation site in human stathmin. Phosphorylation with p34(cdc2)- and cGMP-dependent protein kinases gave almost identical phosphorylation levels in the two stathmins.

Overexpression of Oncoprotein 18 Correlates with Poor Differentiation in Lung Adenocarcinomas

“…Phosphorylated Op18, which increases during mitosis, results in a reduced affinity of Op18 for tubulin and increased microtubule stabilization, thus allowing the mitotic spindle to form (9). Op18 is variably phosphorylated on four distinct serine residues (Ser-16, Ser-25, Ser-38, and Ser-63) in intact cells by different kinases during the cell cycle (10).…”

“…For example, transcription factors (E2F, AP-2, and Sp1) can increase Op18 expression (38), whereas p53 appears to repress the Op18 expression (43). Protein kinases such as cyclin-dependent kinases (CDKs), the cAMP-dependent protein kinase, the mitogen-activated protein/extracellular signal-regulated kinase family, and the Ca 2ϩ /calmodulin-dependent kinase IV/Gr can cause extensive phosphorylation of Op18 protein indicating that Op18 can be influenced by a number of signal transduction pathways (10). We found that the mRNA expression of the transcription factor E2F5 and the protein kinase CDK2 were strongly correlated with Op18 mRNA levels in poorly differentiated bronchial derived adenocarcinomas.…”

Analysis of Cyclin-Dependent Kinase 2-Regulated Phosphorylation of Stathmin in Etoposide-Induced Apoptotic HeLa Cells by Two-Dimensional Electrophoresis and Mass Spectrometry

“…Further, phosphorylation at Ser-25 of stathmin may be due to activation of the MAPK signaling pathway and its phosphorylation may be mediated by MAPK. 26) Consistent with this idea, it has been reported that the MAPK signaling pathway can be significantly activated by some conditions, which include anticancer agent treatment such as etoposide treatment in HeLa cells, 27) while this pathway is constitutively inactivated in JURKAT cells. 28) Collectively, these observations suggest that upregulated expression and increased phosphorylation at Ser-25 of stathmin might be mediated by the MAPK signaling pathway, which is mediated by the cdk2 activation during the onset of etoposide-induced apoptotic events in HeLa cells.…”

L1/Laminin Modulation of Growth Cone Response to EphB Triggers Growth Pauses and Regulates the Microtubule Destabilizing Protein SCG10

“…Anti-phosphopeptide antibodies stained COS-7 cells transfected with wild-type SCG10 (which is known to be phosphorylated in COS cells) but not COS-7 cells transfected with either stathmin or phosphorylation-site mutant forms of SCG10 (Antonsson et al, 1998). In immunoblots, there was no cross-reactivity with the recombinant protein SCG10 produced in Escherichia coli (N-terminally truncated form) or with stathmin expressed in COS-7 cells or purified from E. coli or baculovirus (Antonsson et al, 2001). SCG10 phospho-antibodies were used at 1:50 for immunocytochemistry and at 1:2500 for Western blots.…”