On the cross-regulation of protein tyrosine phosphatases and receptor tyrosine kinases in intracellular signaling

Abstract

Intracellular signaling proteins are very often regulated by site-specific phosphorylation. For example, growth factor receptors in eukaryotic cells contain intrinsic tyrosine kinase activity and use inter- and intra-molecular interactions to recruit and orient potential protein substrates for phosphorylation. Equally important in determining the magnitude and kinetics of such a response is protein dephosphorylation, catalysed by phosphatase enzymes. A growing body of evidence indicates that certain protein tyrosine phosphatases (PTPs), like tyrosine kinases, are affected by intermolecular interactions that alter the specific activity or localization of their catalytic domains. Using a detailed kinetic modeling framework, we theoretically explore the regulation of PTPs through their association with receptor tyrosine kinases, as noted for the Src homology 2-domain-containing PTPs, SHP-1 and -2. Receptor-PTP binding, in turn, is expected to influence the phosphorylation pattern of those receptors and proteins they associate with, and we show how PTPs might serve to co- or counter-regulate parallel pathways in a signaling network.

Introduction

Cells sense the presence of extracellular factors and their concentrations through binding of specific cell surface receptors, which then transduce the stimuli as signals that affect cell function. Within the cell, these signals are often transmitted through protein phosphorylation. Many signaling receptors in eukaryotic cells, notably growth factor receptors, contain intrinsic protein tyrosine kinase (PTK) domains that are activated upon binding of the ligand to their extracellular domains (van der Geer et al., 1994; Schlessinger, 2000), while other signaling receptors must recruit non-receptor tyrosine kinases. Subsequently, the receptors are phosphorylated on multiple tyrosine residues; these phosphorylated residues engage specific intracellular signaling proteins containing modular Src homology 2 (SH2) or phosphotyrosine-binding (PTB) interaction domains (Pawson et al., 2001). An activated receptor thus acts as a scaffold for the assembly of multi-molecular signaling complexes. Many of the signaling proteins that are recruited, either directly or through an adaptor protein, are then phosphorylated by the receptor-associated kinase activity. Protein phosphorylation typically alters the conformation of a protein to affect its function, e.g. its enzymatic and/or intermolecular binding activities, in either a positive or negative manner. Thus, a phosphorylated signaling protein carries the signal initially perceived by the receptor, and the cell maintains many copies of each receptor and signaling protein (typically 10⁴–10⁶ molecules per cell) in order to produce a signal that varies robustly in magnitude as the concentration of an extracellular stimulus is perturbed.

Another important feature of protein phosphorylation is that is readily reversible, such that the fraction of each protein in the phosphorylated state is subject to a dynamic balance of phosphorylation and dephosphorylation rates. In quantitative terms, dephosphorylation allows the intracellular signal to possess a dynamic range of output and to promptly respond to perturbations in the extracellular input(s). Dephosphorylation of phosphotyrosine residues is mediated by protein tyrosine phosphatase (PTP) enzymes. It is now appreciated that a mammalian cell contains many different proteins with classical PTP catalytic domains, each possessing a unique panel of substrate specificities (Tonks and Neel, 2001). Thus, the old view of a few, broad-specificity PTP enzymes has proved to be simplistic. Classical PTP enzymes are further classified as transmembrane (receptor-like) and non-transmembrane (cytosolic) proteins. The catalytic domains of specific PTPs have been found to associate with and act upon receptor PTKs and other phosphorylated signaling proteins, regulating the magnitude and, importantly, the specificity of receptor signaling (Östman and Böhmer, 2001). Interestingly, two classical PTPs, SHP-1 and -2, also contain dual SH2 domains, which allow them to couple directly or indirectly with phosphorylated receptors in the same manner as other signaling proteins. These interactions allosterically activate the PTP catalytic domain by relieving an intramolecular inhibition; perhaps more importantly, the PTP gains better access to receptor-associated substrates (Neel et al., 2003). SHP-1 and -2 are also phosphorylated by receptor-associated PTK activities, which may temporarily lock the PTP in a more active conformation (Feng et al., 1993; Vogel et al., 1993; Zhang et al., 2003). Ironically then, these PTPs seem to use receptor-mediated phosphorylation, in one way or another, to modulate their dephosphorylation activities.

As a relevant example, consider platelet-derived growth factor (PDGF) receptors, which possess intrinsic PTK activity and are self-phosphorylated on at least nine tyrosine sites. These PDGF receptor residues interact with and are dephosphorylated in a site-selective manner by multiple PTPs, including PTP-1B, PTP-PEST, low-molecular weight PTP, the transmembrane PTP DEP-1, as well as the aforementioned SHP-1 and -2 (Klinghoffer and Kazlauskas, 1995; Yu et al., 1998; Kovalenko et al., 2000; Chiarugi et al., 2002; Markova et al., 2003). The SH2 domain(s) of SHP-2 bind phosphorylated Tyr 1009 of the human PDGF β-receptor (Lechleider et al., 1993), enhancing the dephosphorylation of the Tyr 771 receptor site by the SHP-2 catalytic domain (Klinghoffer and Kazlauskas, 1995; Ekman et al., 2002). This receptor site controls the recruitment of RasGAP, which negatively regulates the well-known Ras/Erk cell proliferation pathway, perhaps accounting for the positive effect exerted by SHP-2 on Ras/Erk signaling (Rönnstrand et al., 1999; Zhao and Zhao, 1999). A similar SHP-2-mediated Ras activation mechanism was recently elucidated for epidermal growth factor (EGF) receptor as well (Agazie and Hayman, 2003).

Previous theoretical treatments of protein phosphorylation in models of intracellular signaling have incorporated varying levels of mechanistic detail, depending on the aims and scope of the modeling effort. The simplest model of protein regulation invokes the phosphorylation and dephosphorylation of a single residue, with the latter modeled as a pseudo-first order rate process or with Michaelis-Menten kinetics (Goldbeter and Koshland, 1981; Bhalla and Iyengar, 1999; Brown and Kholodenko, 1999; Kholodenko et al., 1999; Heinrich et al., 2002). Other models have included multiple phosphorylation sites, again with a simplified picture of dephosphorylation activities (Wofsy et al., 1999; Faeder et al., 2003), and yet another described phosphorylation of a single site but accounted for membrane localization effects influencing dephosphorylation mediated by transmembrane versus cytosolic PTPs (Haugh and Lauffenburger, 1998; Haugh et al., 1999). In this paper, we formulate and analyse a multi-site receptor model that explicitly describes the localization and regulation of PTP activities, with an emphasis on PTPs that interact with phosphorylated receptors through both binding and catalytic domains. Our goals are to elucidate signaling phenomena that may result when PTPs are among the signaling proteins recruited by receptors, and to outline possible strategies that may have evolved in the cross-regulation of receptor signaling by PTPs and of PTP activities by receptors.