Identification of Novel Glycogen Synthase Kinase-3β Substrate-interacting Residues Suggests a Common Mechanism for Substrate Recognition

Abstract

Substrate recognition and specificity are essential for the reliability and fidelity of protein kinase function. GSK-3 has a unique substrate specificity that requires prior phosphorylation of its substrates. However, how the enzyme selects its phosphorylated substrates is unknown. Here, we combined in silico modeling with mutagenesis and biological studies to identify GSK-3-substrate interaction sites located within its binding cleft. Protein-protein docking of GSK-3␤ and the phosphorylated cAMP responsive element binding protein (pCREB) (using the available experimentally determined structures), identified Phe 67 , Gln 89 , and Asn 95 of GSK-3␤ as putative binding sites interacting with the CREB phosphorylation motif. Mutations of these residues to alanine impaired GSK-3␤ phosphorylation of several substrates, without abrogating its autocatalytic activity. Subsequently, expression of the GSK-3␤ mutants in cells resulted in decreased phosphorylation of substrates CREB, IRS-1, and ␤-catenin, and prevented their suppression of glycogen synthase activity as compared with cells expressing the wildtype GSK-3␤. Our studies provide important additional understanding of how GSK-3␤ recognizes its substrates: In addition to prior phosphorylation typically required in GSK-3 substrates, substrate recognition involves interactions with GSK-3␤ residues: Phe 67 , Gln 89 , and Asn 95 , which confer a common basis for substrate binding and selectivity, yet allow for substrate diversity. Glycogen synthase kinase 3 (GSK-3)4 is a ubiquitous serine/ threonine kinase expressed as two isoforms (␣ and ␤) (1), and has been implicated in many biological processes, including glucose metabolism, cell apoptosis, and embryonic development (reviewed in Refs. 2-4). The cellular activity of GSK-3 is stringently controlled in response to growth factors and hormones. However, unlike most protein kinases, GSK-3 is constitutively active in resting cells and becomes inhibited upon stimulation of the cells. This inhibition is achieved through direct phosphorylation of N-terminal serine residues (Ser 21 or Ser 9 in ␣, ␤, respectively) by several protein kinases, such as PKB, p90RSK, PKA, and PKC (2-4). GSK-3 also may be phosphorylated on Tyr 216 located in the activation loop (5). This phosphorylation is an autophosphorylation event as demonstrated by in vitro and in vivo cell systems (5-7).Elevated activity of GSK-3 is associated with several diseases, including type 2 diabetes, neurodegenerative diseases, and affective disorders (8 -10). Hence, selective inhibitors of GSK-3 may be of therapeutic value and are currently under extensive development (11)(12)(13)(14). Thus, understanding of how GSK-3 interacts with its substrates may pave the way for design and development of new specific substrate competitive GSK-3 inhibitors.Substrate specificity of protein kinases is a fundamental determinant for the integrity and fidelity of signaling pathways. Previous studies formulated consensus sequences for optimal phosphorylation motifs of protein kinases usi...

Shilianhua extract inhibits GSK-3β and promotes glucose metabolism

“…GSK-3 inhibits insulin signaling by suppression of IRS-1 function through serine phosphorylation (9,10). GSK-3 inhibitors were reported to improve insulin sensitivity in cellular and animal models (2,7,13,14).…”

New Insights into the Autoinhibition Mechanism of Glycogen Synthase Kinase-3β

“…Arg6 forms hydrogen bonds with Gln89 and Asn95, and this is consistent with our previous study that showed the role of these sites in substrate recognition and binding. 22 Arg4 is hydrogen bonded to Asp181, which is highly conserved among all protein kinases and accepts a proton from the hydroxyl group of the substrate. 33 This may explain the inhibitory role of Arg4 as its interaction with Asp181 likely interferes with substrate binding.…”

“…22 The polar nature of Gln89 and Asn95 enables them to form hydrogen bonds with polar/ charged residues in the various substrates. 22 We thus speculated that Gln89 and Asn95 should also play a role in pseudosubstrate binding with the catalytic core. As shown in Fig.…”

“…22 We thus speculated that one or more of these residues may serve as docking sites for the pseudosubstrate. Using molecular, biochemical, and computational analyses, we provide evidence that the pseudosubstrate associates with the active site.…”

The Importance of Being Flexible: The Case of Basic Region Leucine Zipper Transcriptional Regulators

“…GSK-3 recognizes and modifies substrates that are phosphorylated at position +3 with respect to the target phosphorylation site (consensus SXXXpS, where pS denotes phosphorylated serine residue) [173]. Molecular modeling studies [174] indicate that, in the ternary GSK-3, ATP, pKID complex, two helices of pKID are almost collinear. pKID peptide interacts with GSK-3 residues: Phe67, Gln89, and Asn95, whereas the binding pocket for phosphorylated Ser133 consists of side chains of Arg96, Arg180 and Lys205 of GSK-3 (Fig.…”