Characterization of rat brain stathmin isoforms by two-dimensional gel electrophoresis-matrix assisted laser desorption/ionization and electrospray ionization-ion trap mass spectrometry.
Stathmin is a regulatory phosphoprotein that is a target for both cell cycle and cell surface receptor-regulated phosphorylation events. There are at least 14 isoforms of stathmin that migrate on two-dimensional electrophoresis (2-DE): two unphosphorylated, and 12 increasingly phosphorylated proteins. Following extracellular stimuli, stathmin is phosphorylated on four serines (Ser16, Ser25, Ser38, and Ser63) by several kinases, such as mitogen-activated protein (MAP), cdc2 kinase, protein kinase A, and Ca2+/calmodulin-dependent kinase-Gr. While all forms of stathmin are derived from the same protein encoded by a single mRNA, the precise nature of the post-translational modifications has not been clear. In this study we have characterized three rat brain stathmin isoforms, #1, #3 and #4, which electrophorese on 2-DE with apparent molecular weight (Mr)/isoelectric point (pI) values of 15,500/6.2, 15,000/6.1, and 15,000/6.0, respectively. The phosphorylation status of these isoforms was determined using a combination of peptide mapping, matrix-assisted laser desorption/ionization mass spectrometry and electrospray-ionization ion trap mass spectrometry. Stathmin isoform #1 was not phosphorylated, stathmin isoform #3 was phosphorylated on Ser38 only, and stathmin isoform #4 was phosphorylated on Ser38; however, the phosphorylation status of Ser63 could not be determined. In addition, three proteins which electrophorese near stathmin were identified in order to more accurately define the Mr/pI locus of this region of the 2-DE gel map. These include: phosphatidyl ethanolamine binding protein (Mr approximately 18,000/pI 6.0), synuclein forms 2 and 3 (Mr approximately 14,000/pI 5.4), and synuclein form 2 (Mr approximately 15,000/pI 5.0).
Zugaro
L M
LM
Joint Protein Structure Laboratory, Ludwig Institute for Cancer Research (Melbourne Branch) and the Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia.
Reid
G E
GE
Ji
H
H
Eddes
J S
JS
Murphy
A C
AC
Burgess
A W
AW
Simpson
R J
RJ
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001923:Brain Chemistry; D015180:Electrophoresis, Gel, Two-Dimensional; D007536:Isomerism; D008868:Microtubule Proteins; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000737:chemistry*; D010766:Phosphorylation; D051381:Rats; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D050777:Stathmin
D008868:Microtubule Proteins; D010750:Phosphoproteins; D050777:Stathmin