Volume 272, Number 18, Issue of May 2, 1997 pp. 12164-12169
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

---

Proteasomal Degradation of Spermidine/Spermine N¹-Acetyltransferase Requires the Carboxyl-terminal Glutamic Acid Residues

(Received for publication, January 17, 1997, and in revised form, February 24 1997)

From the Department of Cellular and Molecular Physiology, The Milton S. Hershey Medical Center, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033

The rapid turnover of spermidine/spermine N¹-acetyltransferase (SSAT), a key enzyme in the regulation of polyamine levels, was found to be mediated via ubiquitination and the proteasomal system. SSAT degradation was blocked by the binding of polyamines or of the polyamine analog, N¹,N¹²-bis(ethyl)spermine (BE-3-4-3), to the protein, providing a mechanism for the increase of SSAT activity in response to these agents. Site-directed mutagenesis indicated that a number of residues including arginine 19, cysteine 122, histidine 126, glutamic acid 152, arginine 155, and methionine 167 were needed for protection of SSAT by BE-3-4-3. These residues have previously been shown to reduce the affinity for the binding of polyamines to the SSAT protein, and these results indicate that the change in protein configuration brought about by this binding renders the protein resistant to proteasomal degradation. Mutations to alanines of residues arginine 7, cysteine 14, and lysine 141 also prevented the protection by BE-3-4-3, and these residues may be required for the formation of the protected conformation. The rapid degradation of SSAT required the carboxyl-terminal region of the protein, and the two terminal glutamic acid residues at positions 170 and 171 were found to be of critical importance. Truncation of the protein to remove these residues or the mutation of either of these acidic residues to glutamine completely abolished the rapid degradation of SSAT. The addition of two extra lysine residues at the carboxyl terminus or the conversion of the glutamic acids at positions 170 and 171 to lysines also prevented SSAT degradation by the proteasome. These results show the key role of the acidic residues at the carboxyl terminus of the protein in reacting with the proteasome. In contrast, mutation of lysine 166 to alanine, which extends the length of the acidic region in the carboxyl-terminal fragment of SSAT, actually increased the rate of degradation of SSAT without affecting its stabilization by BE-3-4-3. The binding of BE-3-4-3 or polyamines is therefore likely to change the configuration of the SSAT protein in a way that prevents the exposure of the carboxyl-terminal region of the ubiquitinated protein to the proteasome.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				A. E. Pegg Spermidine/spermine-N1-acetyltransferase: a key metabolic regulator Am J Physiol Endocrinol Metab, June 1, 2008; 294(6): E995 - E1010. [Abstract] [Full Text] [PDF]

				N. J. Butcher, G. M. Broadhurst, and R. F. Minchin Polyamine-dependent Regulation of Spermidine-Spermine N1-Acetyltransferase mRNA Translation J. Biol. Chem., September 28, 2007; 282(39): 28530 - 28539. [Abstract] [Full Text] [PDF]

				K. Zahedi, J. J. Bissler, Z. Wang, A. Josyula, L. Lu, P. Diegelman, N. Kisiel, C. W. Porter, and M. Soleimani Spermidine/spermine N1-acetyltransferase overexpression in kidney epithelial cells disrupts polyamine homeostasis, leads to DNA damage, and causes G2 arrest Am J Physiol Cell Physiol, March 1, 2007; 292(3): C1204 - C1215. [Abstract] [Full Text] [PDF]

				M. C. Bewley, V. Graziano, J. Jiang, E. Matz, F. W. Studier, A. E. Pegg, C. S. Coleman, and J. M. Flanagan Structures of wild-type and mutant human spermidine/spermine N1-acetyltransferase, a potential therapeutic drug target PNAS, February 14, 2006; 103(7): 2063 - 2068. [Abstract] [Full Text] [PDF]

				D. E. McCloskey and A. E. Pegg Properties of the Spermidine/Spermine N1-Acetyltransferase Mutant L156F That Decreases Cellular Sensitivity to the Polyamine Analogue N1, N11-Bis(ethyl)norspermine J. Biol. Chem., April 11, 2003; 278(16): 13881 - 13887. [Abstract] [Full Text] [PDF]

				C. S. Coleman, A. E. Pegg, L. C. Megosh, Y. Guo, J. A. Sawicki, and T. G. O'Brien Targeted expression of spermidine/spermine N1-acetyltransferase increases susceptibility to chemically induced skin carcinogenesis Carcinogenesis, February 1, 2002; 23(2): 359 - 364. [Abstract] [Full Text] [PDF]

				Y. Wang, W. Devereux, P. M. Woster, T. M. Stewart, A. Hacker, and R. A. Casero Jr. Cloning and Characterization of a Human Polyamine Oxidase That Is Inducible by Polyamine Analogue Exposure Cancer Res., July 1, 2001; 61(14): 5370 - 5373. [Abstract] [Full Text] [PDF]

				I. P. Ivanov, R. F. Gesteland, and J. F. Atkins SURVEY AND SUMMARY: Antizyme expression: a subversion of triplet decoding, which is remarkably conserved by evolution, is a sensor for an autoregulatory circuit Nucleic Acids Res., September 1, 2000; 28(17): 3185 - 3196. [Abstract] [Full Text] [PDF]

				P. Coffino Polyamines in spermiogenesis: Not now, darling PNAS, April 25, 2000; 97(9): 4421 - 4423. [Full Text] [PDF]

				R. K. Mallampalli, A. J. Ryan, R. G. Salome, and S. Jackowski Tumor Necrosis Factor-alpha Inhibits Expression of CTP:Phosphocholine Cytidylyltransferase J. Biol. Chem., March 24, 2000; 275(13): 9699 - 9708. [Abstract] [Full Text] [PDF]

				K. Su, M. D. Roos, X. Yang, I. Han, A. J. Paterson, and J. E. Kudlow An N-terminal Region of Sp1 Targets Its Proteasome-dependent Degradation in Vitro J. Biol. Chem., May 21, 1999; 274(21): 15194 - 15202. [Abstract] [Full Text] [PDF]

				D. E. McCloskey, C. S. Coleman, and A. E. Pegg Properties and Regulation of Human Spermidine/Spermine N1-Acetyltransferase Stably Expressed in Chinese Hamster Ovary Cells J. Biol. Chem., March 5, 1999; 274(10): 6175 - 6182. [Abstract] [Full Text] [PDF]

				A. Schmidt, R. Grimm, J. Schmidt, D. Scheel, D. Strack, and S. Rosahl Cloning and Expression of a Potato cDNA Encoding Hydroxycinnamoyl-CoA:Tyramine N-(Hydroxycinnamoyl)transferase J. Biol. Chem., February 12, 1999; 274(7): 4273 - 4280. [Abstract] [Full Text] [PDF]