Identification of Plectin as a Substrate of p34
Kinase and Mapping of a Single Phosphorylation Site (*)
- From the Institute of Biochemistry and Molecular Cell Biology, University of Vienna, Biocenter, Dr. Bohrgasse 9, A-1030 Vienna, Austria
Abstract
Plectin is an in vitro substrate for various kinases present in cell lysates from mitotic and interphase Chinese hamster ovary cells. Sensitivity
of plectin kinase activity to the inhibitor olomoucine, and two-dimensional tryptic peptide mapping of plectin phosphorylated
by various kinase preparations suggested that the major plectin kinase activity in mitotic extracts is related to the cell
cycle regulator kinase p34
. Bacterial expression of various truncated plectin mutant proteins comprising different domains of the molecule and their
phosphorylation by purified p34
kinase revealed that the target site of this kinase resided within plectin's C-terminal globular domain. Among the subdomains
of the C-terminal region (six repeats and a short tail sequence), only repeat 6 and the tail were phosphorylated by p34
kinase. As shown by two-dimensional phosphopeptide mapping, repeat 6, but not the tail, contained a mitosis-specific phosphorylation
site targeted by p34
kinase in intact plectin molecules. By performing site-directed mutagenesis of a potential p34
recognition sequence motif within the repeat 6 domain, threonine 4542 was identified as the major target for the kinase.
Protein kinase A, phosphorylating plectin also within repeat 6, targeted sites that were clearly different from those of p34
kinase.
Footnotes
-
↵* This study was supported by grants from the Austrian Science Research Fund (to R. F. and G. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
-
↵1 The abbreviations used are:
- IF
-
intermediate filament
- MBP
-
maltose-binding protein
- DMEM
-
Dulbecco's modified Eagle's medium
- FCS
-
fetal calf serum
- S-MEM
-
Joklik's modified minimum essential medium
- PMSF
-
phenylmethylsulfonyl fluoride
- PCR
-
polymerase chain reaction
- RIPA
-
radioimmune precipitation buffer
- PAGE
-
polyacrylamide gel electrophoresis
- bp
-
base pair(s).
-
- Received November 15, 1995.
- Revision received January 18, 1996.
- © 1996 by The American Society for Biochemistry and Molecular Biology, Inc.
