Enzyme Is the Name-Adapter Is the Game

Abstract

Signaling proteins in eukaryotes usually comprise a catalytic domain coupled to one or several interaction domains, such as SH2 and SH3 domains. An additional class of proteins critically involved in cellular communication are adapter or scaffold proteins, which fulfill their purely non-enzymatic functions by organizing protein-protein interactions. Intriguingly, certain signaling enzymes, e.g., kinases and phosphatases, have been demonstrated to promote particular cellular functions by means of their interaction domains only. In this review, we will refer to such a function as "the adapter function of an enzyme". Though many stories can be told, we will concentrate on several proteins executing critical adapter functions in cells of the immune system, such as Bruton´s tyrosine kinase (BTK), phosphatidylinositol 3-kinase (PI3K), and SH2-containing inositol phosphatase 1 (SHIP1), as well as in cancer cells, such as proteins of the rat sarcoma/extracellular signal-regulated kinase (RAS/ERK) mitogen-activated protein kinase (MAPK) pathway. We will also discuss how these adaptor functions of enzymes determine or even undermine the efficacy of targeted therapy compounds, such as ATP-competitive kinase inhibitors. Thereby, we are highlighting the need to develop pharmacological approaches, such as proteolysis-targeting chimeras (PROTACs), that eliminate the entire protein, and thus both enzymatic and adapter functions of the signaling protein. We also review how genetic knock-out and knock-in approaches can be leveraged to identify adaptor functions of signaling proteins.

Keywords: ErbB3/HER3; KSR1; SHP2; cancer; dimerization; immunodeficiency; inflammatory diseases; protein complexes; pseudokinase.

Figure 1

Differentiating between enzymatic function and adapter function. (A) An enzyme (in this example, we show a kinase) consisting of interaction domains (ID) and a kinase domain (KD) interacts with protein X in a way that allows the kinase to phosphorylate protein X and modulate its function. We are using the term “enzymatic function” for such interactions. (B) The kinase via one of its IDs connects protein X and protein Y, thus allowing these proteins to functionally interact (bilateral arrow). In this example, the enzymatic activity (KD) of the kinase is not relevant for this functional interaction. In such a scenario, we use the term “adapter function”. (C) In the situation shown, the kinase interacts with the plasma membrane (PM) or an organelle. Using one of its IDs, the kinase promotes the functional interaction between protein Z and the membrane. In this scenario, the KD of the kinase is not relevant for the function of protein Z (arrow), and we use the term “adapter function” for such situations.

Figure 2

BTK structure and catalytic as well as adapter functions. BTK comprises (from N- to C-terminus) a PH-domain, a TH-domain, an SH3-domain, an SH2-domain, as well as a tyrosine kinase (TK) domain (also known as SH1-domain). The following amino acids are accentuated: Y551 has to be phosphorylated for BTK activation; Y223 can be auto-phosphorylated by active BTK; C481 in the catalytic domain can form a covalent bond with the inhibitor Ibrutinib; and R28 within the PH-domain is crucial for PIP₃ interaction. The red question mark alludes to the fact that in certain cells upon differential stimulation, BTK activation does not appear to be dependent on PI3K activation (see text). One dominant catalytic function (highlighted by a red dashed rectangle) is the phosphorylation/activation of PLCγ1/2, which then hydrolyzes PI45P₂ to yield IP₃ and DAG to promote signaling towards Ca²⁺ mobilization and PKC activation, respectively. The adapter functions known so far (highlighted by green dashed rectangles) comprise the SH2-mediated interaction with the adapter protein SLP65, which together execute a tumor suppressing function in the B-cell lineage, as well as the interaction of PIP5K with BTK’s PH-domain allowing PIP5K translocation to the plasma membrane to phosphorylate the phospholipid PI4P to yield PI45P₂. BTK-promoted PI45P₂ production is thought to constitute two feed-forward loops to provide the substrate for both PLCγ1/2 and PI3K.

Figure 3

SHIP1 structure and catalytic as well as adapter functions. SHIP1 comprises (from N- to C-terminus) an SH2-domain, a catalytic 5′-phosphatase (PPtase) domain containing a PI34P₂-binding C2 domain allowing allosteric feed-forward enhancement, and a proline-rich (P) C-terminus additionally comprising two NPxY sequences (Y912 and Y1020). As an enzyme, SHIP1 hydrolyzes the phospholipid PIP3 to yield PI34P₂ (highlighted by a red dashed rectangle). The adapter functions reported so far (highlighted by green dashed rectangles) comprise the SH2-mediated displacement of the GRB2-SOS complex from the adapter protein SHC, thus repressing GDP-to-GTP exchange at RAS (resulting in enhanced RAS^GDP to RAS^GTP levels (RAS^GDP > RAS^GTP)) as well as the recruitment of the DOK1-RASGAP1 complex to a C-terminal phosphorylated NPxY sequence via DOK1´s PTB-domain, hence causing enhanced GTP-to-GDP hydrolysis at RAS (RAS^GDP > RAS^GTP). Moreover, the E3 ubiquitin ligase XIAP can interact with the proline-rich tail of SHIP1, thereby displacing XIAP from RIP2, and blocking NFκB activation. Furthermore, the SH2-domain of SHIP1 has been demonstrated to interact with phosphorylated Y1020, allowing dimerization (and most probably oligomerization) of SHIP1 to promote its potential scaffolding function.

Figure 4

Simplified sketch of the RAS/ERK MAPK pathway. This pathway is activated by a plethora of receptor tyrosine kinases (RTK), such as the subsequently mentioned HER2/HER3 heterodimer, but also antigen and cytokine receptors [61,62,63]. Phosphorylation of the receptor tails, either by intrinsic enzymatic activity as in the case of RTKs, or by associated protein tyrosine kinases of the SRC, SYK, or JAK families in case of antigen and cytokine receptors, results in the recruitment of SH2 domain containing adaptor proteins like GRB2. With its two SH3 domains, GRB2 can then recruit the guanine nucleotide exchange factor SOS and docking proteins of the GAB family, which, upon tyrosine phosphorylation, recruit and allosterically activate SHP2 by engaging with its tandem SH2 domain [64,65]. Both SOS and active SHP2 are required for optimal RAS signaling. Active RAS not only recruits RAF family members, such as BRAF and RAF1 to the plasma membrane, but also induces conformational changes resulting in the exposure of their kinase domains and homo- or heterodimerization, leading to the allosteric transactivation of RAF protomers indicated by the swung red arrow [66,67,68]. Likewise, the pseudokinase KSR1 or RAF proteins rendered inactive either by mutations abolishing enzymatic activity or by ATP competitive kinase inhibitors can also trigger allosteric transactivation of catalytically competent RAFs [69,70]. Figure was created with BioRender.com (accessed on 27 April 2024).