ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 272, No. 2, August 1, pp. 274-280,1989 Substrate Phosphorylation Can Inhibit Proteolysis by Trypsin-like Enzymes’ MARILEE BENORE-PARSONS, NABIL G. SEIDAH,* AND LAWRENCE P. WENNOGLE’ Research Department, C&a-Geigy Corporation, Summit, New Jersey 07901, and *Laboratm of Biochemical Neuroendocrinology, Clinical Research Institute of Mmtreal, 110 Pine Avenue West, Montreal, P.Q., Canada H3E lK7 Received November 21,1988, and in revised form March 16,1989 The effect of substrate phosphorylation on the susceptibility to proteolytic cleavage by trypsin-like enzymes was investigated using the model heptapeptide Leu-Arg-Arg- Ala-Ser-Leu-Gly, a peptide representing the endogenous phosphorylation site of pyru- vate kinase. Phosphorylation of Ser 5 altered the kinetics of proteolysis by two proteases, trypsin and rat plasma kallikrein, both of which cleaved between Arg 3 and Ala 4. In the case of trypsin, phosphorylation decreased the rate of cleavage 47-fold. In the case of rat plasma kallikrein, phosphorylation decreased proteolysis l&fold. Phosphorylation resulted in an apparent redirection of the preferential site from Arg 3 to Arg 2. Because sequences analogous to this model peptide are commonly found in exposed domains of globular proteins, and since these regions are susceptible to both phosphorylation and protease attack, the results indicate that substrate phosphorylation may selectively in- fluence protein processing and turnover. a 1989 Academic Press, rnc. Protein processing and turnover is criti- cal to cellular homeostasis and adaptation, and reflecting this importance the turn- over rates of different eukaryotic proteins vary tremendously (1). A number of sys- tems are known to mediate the turnover of intracellular proteins, such as the ubiqui- tin pathway (2) and the receptor-mediated endocytotic systems (3,4). Recent theories indicate that certain primary amino acid sequences act as signals which specify rates of degradation (5,6). Extracellularly, both proteolytic and nonproteolytic mech- anisms contribute to protein turnover and clearance. A number of proteases are involved in protein metabolism, including families of metallo, cysteine, aspartic, and serine pep- 1 Portions of this work were presented at the 1987 ASBC confereace (Benore-Parsons, M., and Wen- nogle, L. P. (1987) Fed Proc. 46,2086). ‘To whom correspondence should be addressed. tidases. Notable among the intracellular proteases are the lysosomal and calcium- activated enzymes (7, 8). An additional class of proteases has recently been docu- mented, referred to either as “basic cleav- age enzymes” or as “trypsin-like en- zymes,” a family of proteases involved in protein processing (9,10). We have looked for alternate mechanisms which might modulate protein turnover by this class of proteases, and focused on protease-sensi- tive dibasic amino acid sequences adjacent to phosphorylation sites. Such sequences are often found at turns or exposed regions of proteins (11, 12), and consensus se- quences for cyclic adenosine monophos- phate, cyclic guanosine monophosphate, calmodulin, and calcium/phospholipid de- pendent kinases all contain basic residues in proximity to target serine and threonine residues (13,14). This paper describes the use of a model peptide to study the effect of substrate 0003-9861/89 $3.00 Copyright 0 1989 by Academic Press. Inc. All rights of reproduction in any form reserved. 274