Multifaceted interactions and regulation between antizyme and its interacting proteins cyclin D1, ornithine decarboxylase and antizyme inhibitor

Abstract

Ornithine decarboxylase (ODC), cyclin D1 (CCND1) and antizyme inhibitor (AZI) promote cell growth. ODC and CCND1 can be degraded through antizyme (AZ)-mediated 26S proteasomal degradation. This paper describes a mechanistic study of the molecular interactions between AZ and its interacting proteins. The dissociation constant (Kd) of the binary AZ-CCND1 complex and the respective binding sites of AZ and CCND1 were determined. Our data indicate that CCND1 has a 4-fold lower binding affinity for AZ than does ODC and an approximately 40-fold lower binding affinity for AZ than does AZI. The Kd values of AZ-CCND1, AZ-ODC and AZ-AZI were 0.81, 0.21 and 0.02 μM, respectively. Furthermore, the Kd values for CCND1 binding to the AZ N-terminal peptide (AZ34-124) and AZ C-terminal peptide (AZ100-228) were 0.92 and 8.97 μM, respectively, indicating that the binding site of CCND1 may reside at the N-terminus of AZ, rather than the C-terminus. Our data also show that the ODC-AZ-CCND1 ternary complex may exist in equilibrium. The Kd values of the [AZ-CCND1]-ODC and [AZ-ODC]-CCND1 complexes were 1.26 and 4.93 μM, respectively. This is the first paper to report the reciprocal regulation of CCND1 and ODC through AZ-dependent 26S proteasomal degradation.

Keywords: biochemistry; cell cycle; molecular and cellular biology; oncogene; signal transduction.

Figure 1. Continuous sedimentation coefficient distribution of the human CCND1 protein and the AZ-CCND1 complex

A. Three concentrations of the CCND1 protein in 30 mM Tris-HCl buffer (pH 7.4) at 20°C were used in the experiment: 0.05, 0.1 and 0.3 mg/ml. The sedimentation velocity data were globally fitted using the SEDPHAT program to obtain the K_d of the CCND1 dimer (Table 1). B. The concentration of AZ was fixed at 0.25 mg/ml, and CCND1 concentrations of 0.19, 0.37 and 0.74 mg/ml (the molar ratios of CCND1/AZ were 0.5, 1 and 2, respectively) in a buffer containing 30 mM Tris-HCl (pH 7.4) and 50 mM NaCl were used. The sedimentation velocity data were globally fitted using SEDPHAT to obtain the K_d of the AZ-CCND1 complex (Table 1).

Figure 2. Interactions between the N-terminal and the C-terminal domains of human AZ and CCND1

The protein concentrations of AZ, AZ_34–124, AZ_100–228, CCND1, CCND1_21–154 and CCND1_155–295 were 0.25, 0.075, 0.077, 0.17, 0.4 and 0.35 mg/ml, respectively, in a buffer containing 30 mM Tris-HCl (pH 7.4) and 50 mM NaCl. The sedimentation velocity was determined at 20°C, and the molar ratio of AZ/CCND1 was fixed at 1.

Figure 3. Continuous sedimentation coefficient distribution of human CCND1 in the presence of AZ_34–124 and AZ_100–228

The concentrations of AZ_34–124 and AZ_100–228 were fixed at 0.1 and 0.25 mg/ml, respectively, whereas CCND1 concentrations of 0.12, 0.25 and 0.37 mg/ml for AZ_34–124 and of 0.17, 0.35 and 0.52 mg/ml for AZ_100–228 (corresponding to molar ratios of CCND1/AZ of 0.5, 1 and 1.5, respectively) in a buffer containing 30 mM Tris-HCl (pH 7.4) and 50 mM NaCl were used. The sedimentation velocity data were globally fitted using SEDPHAT to obtain the K_d of the AZ-CCND1 complex (Table 1).

Figure 4. Continuous sedimentation coefficient distribution of AZ with its interacting proteins, ODC, CCND1 and AZI

The protein concentrations of AZ, ODC, CCND1 and AZI were 0.2, 0.48, 0.3 and 0.47 mg/ml, respectively, in a buffer containing 30 mM Tris-HCl (pH 7.4) and 50 mM NaCl. The sedimentation velocity was determined at 20°C, and the molar ratio of AZ to each interacting protein was fixed at 1.

Figure 5. Continuous sedimentation coefficient distributions of the AZ-ODC complex with increasing concentrations of CCND1 and of the AZ-CCND1 complex with increasing concentrations of ODC

The sedimentation velocity was determined at 20°C, and the proteins were diluted in a buffer containing 30 mM Tris-HCl (pH 7.4) and 50 mM NaCl. A. The molar ratio of AZ/ODC was fixed at 1, and the CCND1 protein concentration was varied. B. The molar ratio of AZ/CCND1 was fixed at 1, and the ODC protein concentration was varied.

Figure 6. AZ-mediated ODC and CCND1 protein degradation

The degradation of recombinant ODC or CCND1 protein in the presence of AZ was examined by reticulocyte lysate-based in vitro degradation. Human anti-ODC and anti-CCND1 antibodies were utilized as probes in subsequent immunoblotting experiments. A. AZ-mediated ODC degradation. Lanes 1 and 2: ODC only. Lanes 3 and 4: ODC mixed with AZ; the molar ratio of [ODC]/[AZ] was 1:1. Lanes 5 and 6: ODC-AZ complex mixed with CCND1; the molar ratio of [ODC]/[AZ]/[CCND1] was 1:1:5. B. AZ-mediated CCND1 degradation. Lanes 1 and 2: CCND1 only. Lanes 3 and 4: CCND1 mixed with AZ; the molar ratio of [CCND1]/[AZ] was 1:3. Lanes 5 and 6: CCND1-AZ complex mixed with ODC; the molar ratio of [ODC]/[AZ]/[CCND1] was 1:3:1.

Figure 7. Molecular interactions between AZ and its interacting proteins

A. Binding elements within AZ and CCND1. B. Mode of AZ binding to its interacting proteins.