Identification of Plectin as a Substrate of p34 Kinase and Mapping of a Single Phosphorylation Site

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 cdc2. Bacterial expression of various truncated plectin mutant proteins comprising different domains of the molecule and their phosphorylation by purified p34 cdc2 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 cdc2 kinase. As shown by two-dimensional phosphopeptide mapping, repeat 6, but not the tail, contained a mitosis-specific phosphorylation site targeted by p34 cdc2 kinase in intact plectin molecules. By performing site-directed mutagenesis of a potential p34 cdc2 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 cdc2 kinase.Plectin is an abundant cytoskeletal protein of exceptionally large size. Electron microscopy of purified plectin molecules (1) and structure prediction based on the cloning and sequencing of rat plectin cDNA (2) revealed an extended central rod and two flanking globular domains as distinctive structural features. Its subcellular distribution, in particular its partial codistribution with intermediate filaments (IFs) 1 and prominent occurrence at plasma membrane attachment sites of IFs and microfilaments, and the identification of numerous specific binding proteins at the molecular level (reviewed in Refs. 3 and 4) suggested that plectin might be involved in versatile cytoplasmic cross-linking functions. In a first approach to characterize plectin's various binding domains, transient transfection of mammalian cells using cDNAs encoding plectin mutant proteins indicated a role of the C-terminal globular domain in the binding to vimentin (5).As a prominent phosphoprotein plectin was found to be an in vivo target of a Ca 2ϩ /calmodulin-dependent kinase and of protein kinases A and C (6 -8). In vitro studies demonstrated that plectin's capacity to bind to IF proteins, such as vimentin and lamin B, were differentially influenced by phosphorylation (8), suggesting that distinct protein kinases were involved in regulating at least some of plectin's interactions.In view of plectin's proposed role as a cytoplasmic crosslinking element, a specific regulation of its binding activities would seem of particular importance during mitosis, when dramatic structural rearrangements of the cytoskeleton, including IF networks, take place. In fact, two of plectin's well characterized binding partners, vi...

Naringin-sensitive Phosphorylation of Plectin, a Cytoskeletal Cross-linking Protein, in Isolated Rat Hepatocytes

“…For example, plectin can be phosphorylated at Thr 4542 by the cyclin-dependent protein kinase CDK1/Cdc2 during mitosis, causing it to dissociate from vimentin filaments (64,65). Since plectin phosphorylation in the present study was detected by an anti-phosphothreonine antibody, it is clear that the toxin-sensitive phosphorylation site must likewise be a threonine.…”

BRCA2 interacts with the cytoskeletal linker protein plectin to form a complex controlling centrosome localization

“…(27) Furthermore, the IF-binding site of plectin has been located between PLEC M5 and M6 (18) ; in addition, the carboxyterminal M6 contains a unique cell cycle kinase cdk1 phosphorylation site. (25) So, reflecting on the possibility that BRCA2 interacts with any of the plectin modules, we took notice of PLEC M1 (2738-3021 a.a) and the C-terminal region (4150-4503 a.a) (which comprised PLEC M6, along with an adjacent vimentin site; GST-PLEC M1 and GST were incubated with BRCA2-FLAG or FLAG, and a pull-down assay was then performed. Lane 1, GST incubated with FLAG; lane 2, GST incubated with BRCA2-FLAG; lane 3, GST-PLEC M1 incubated with FLAG; lane 4, GST-PLEC M1 incubated with BRCA2-FLAG.…”

Fbx7 Functions in the SCF Complex Regulating Cdk1-Cyclin B-phosphorylated Hepatoma Up-regulated Protein (HURP) Proteolysis by a Proline-rich Region

“…3B). Second, Cdk1-specific inhibitors, roscovitine (33) and olomoucine (34), partially abolished the mobility up-shifts of HURP as [ 35 S]methionine-labeled HURP was incubated with nocodazole-arrested 293T lysates (Fig. 3C), suggesting that HURP is a potential substrate of Cdk1-cyclin B in the G 2 /M phase.…”