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. 2000 Nov 7;97(23):12589-94.
doi: 10.1073/pnas.220423497.

Peripheral Golgi protein GRASP65 is a target of mitotic polo-like kinase (Plk) and Cdc2

C Y Lin  1 , M L Madsen, F R Yarm, Y J Jang, X Liu, R L Erikson
Affiliations

Peripheral Golgi protein GRASP65 is a target of mitotic polo-like kinase (Plk) and Cdc2

C Y Lin et al. Proc Natl Acad Sci U S A. .

Abstract

Cell division is characterized by orchestrated events of chromosome segregation, distribution of cellular organelles, and the eventual partitioning and separation of the two daughter cells. Mitotic kinases, including polo-like kinases (Plk), influence multiple events in mitosis. In yeast two-hybrid screens using mammalian Plk C-terminal domain baits, we have identified Golgi peripheral protein GRASP65 (Golgi reassembly stacking protein of 65 kDa) as a Plk-binding protein. GRASP65 appears to function in the postmitotic reassembly of Golgi stacks. In this report we demonstrate binding between Plk and GRASP65 and provide in vitro and in vivo evidence that Plk is a GRASP65 kinase. Moreover, we show that Cdc2 can also phosphorylate GRASP65. In addition, we present data which support the observation that the conserved C terminus of Plk is important for its function. Deletion or frameshift mutations in the conserved C-terminal domain of Plk greatly diminish its ability to phosphorylate GRASP65. These and previous findings suggest that phosphorylation of Golgi components by mitotic kinases may regulate mechanisms of Golgi inheritance during cell division.

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Figures

Figure 1
Figure 1
Yeast two-hybrid interactions between GRASP65 (amino acids 7–120) and Plk. Conserved polo-boxes (1–3, left to right) are indicated by darkly shaded boxes. Binding interaction between Plk and GRASP65 was determined by growth on selective medium and β-galactosidase activity. Data are not available for the full-length and 323–603 constructs because of background β-galactosidase activity in bait strains.
Figure 2
Figure 2
Expression and characterization of epitope-tagged GRASP65. (A) Full-length GRASP65 was cloned from rat liver cDNA and a C-terminal FLAG epitope tag was added. A 1-μg sample of vector (pBS), WT (M+), or myristoylation site mutant (M−) constructs was added to each in vitro expression reaction mixture. Products were labeled with Tran35S-label (ICN) for visualization and immunoprecipitated (IP) with anti-FLAG antibodies to determine the integrity of the epitope tag. GRASP65-FLAG is indicated by arrows. (B) Cos-7 cells transfected with GRASP65-FLAG were arrested in mitosis or allowed to divide randomly and labeled with [32P]orthophosphate for 3 h. Phosphoproteins were immunoprecipitated, resolved by SDS/PAGE, and visualized by autoradiography. Multiple phosphorylated bands are indicated by arrows.
Figure 3
Figure 3
GRASP65 coimmunoprecipitates with Plk. (A) Anti-FLAG immunoprecipitations (IP). HA-Plk is indicated by arrow. (B) Anti-HA immunoprecipitations. GRASP65-FLAG is indicated by arrow. Fusion proteins were detected by immunoblotting (IB) with their respective epitope antibodies. Cos-7 cells were transfected with vector (pCIneo) only (lane 1) or cotransfected with GRASP65-FLAG and HA-Plk WT (lane 2), KD (lane 3), polo-box 1 (a.a. 411–439) FS (lane 4), or T210D (lane 5) constructs at a 1:3 ratio.
Figure 4
Figure 4
Plk and Cdc2 are GRASP65 kinases. (A) Plk is a GRASP65 kinase. GRASP65-FLAG immunoprecipitated from in vitro expression reactions (lanes 1–4) or from transfected Cos-7 cells (lanes 5–8) was incubated with soluble GST-Plk (KD or WT) in kinase reaction buffer. A 0.5 μg sample of either vector (pBS) or GRASP65-FLAG constructs was used in each expression reaction. Equal aliquots of immunoprecipitates from transfected cells were used in each kinase reaction. (B) Expression of Plk enhances in vivo phosphorylation of GRASP65. Cos-7 cells were cotransfected with pCIneo vector (lanes 1 and 2) or HA-Plk WT (lanes 3–5), KD (lanes 6–8) or T210D (lanes 9–11), and GRASP65-FLAG. In each cotransfection experiment, 2.5, 5, or 7.5 μg of Plk constructs was used. Vector-only controls (lanes 1 and 2) were either treated with nocodazole (Mit.) or left untreated (C) to determine basal levels of GRASP65 phosphorylation. Transfected cells were incubated with [32P]orthophosphate for 3 h in phosphate-free medium, and GRASP65 was isolated by anti-FLAG immunoprecipitations (IP) and resolved by SDS/PAGE. Phosphorylated GRASP65 was detected by autoradiography (Upper), and the amount of GRASP65 in the immunocomplex was determined by Western analysis with anti-FLAG antibody (Lower). (C) GRASP65 is also phosphorylated by Cdc2. GRASP65-FLAG immunoprecipitated from in vitro expression reactions was incubated with soluble GST-Plk (KD or WT) or GST-cyclin B-Cdc2 in kinase reaction buffer.
Figure 5
Figure 5
Tryptic mapping of phosphorylated GRASP65. GRASP65-FLAG immunoprecipitated from in vitro expression reactions was labeled by purified GST-PlkWT (Upper Right), GST-cyclin B-Cdc2 (Lower Left), and radiolabeled by endogenous kinases (Upper Left) in transfected cells arrested in mitosis by nocodazole treatment and was recovered by immunoprecipitation. Phosphoproteins were eluted from the gel after SDS/PAGE and subjected to tryptic digest. Digestion products were separated in two dimensions by electrophoresis (pH 1.9) and chromatography and detected by autoradiography. Nine major phosphopeptides (labeled 1–9) were detected in a mixture (Lower Right) of in vitro and in vivo products. Plk and Cdc2 phosphorylate specific sites within GRASP65 that are relevant in vivo. The additional phosphopeptide (peptide 7) in the GRASP65 labeled in vivo suggests the existence of at least one more GRASP65 kinase other than Plk and Cdc2.
Figure 6
Figure 6
The conserved C-terminal domain of Plk is important for GRASP65 phosphorylation. (A) Deletion of conserved Plk C terminus (amino acids 356–603 deleted) diminishes GRASP65 phosphorylation (lane 5). Comparable casein kinase activity was present in the WT or ΔC reactions (Left). GRASP65-FLAG expressed in vitro was immunoprecipitated and incubated in kinase reaction buffer with purified GST-PlkKD, WT, or ΔC. (B) HA-Plk polo-box 1 FS mutant coimmunoprecipitates with GRASP65-FLAG but does not efficiently phosphorylate it in an immunocomplex kinase assay (lane 9). The immunocomplexes were denatured after the kinase reaction was terminated, and GRASP65-FLAG was immunoprecipitated with anti-FLAG antibodies and subjected to autoradiography. The background kinase activity observed in KD (lane 8) and FS immunocomplexes is partly due to coimmunoprecipitating endogenous kinases (see GRASP65-FLAG-only control, lane 2).
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