Current thoughts on cellular functions of numb-associated kinases

Abstract

Members of the Numb-associated kinase family of serine/threonine kinases play an essential role in many cellular processes, such as endocytosis, autophagy, dendrite morphogenesis, osteoblast differentiation, and the regulation of the Notch pathway. Numb-associated kinases have been relevant to diverse diseases, including neuropathic pain, Parkinson’s disease, and prostate cancer. Therefore, they are considered potential therapeutic targets. In addition, it is reported that Numb-associated kinases have been involved in the life cycle of multiple viruses such as hepatitis C virus (HCV), Ebola virus (EBOV), and dengue virus (DENV). Recently, Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to threaten global health. Studies show that Numb-associated kinases are implicated in the infection of SARS-CoV-2 which can be suppressed by Numb-associated kinases inhibitors. Thus, Numb-associated kinases are proposed as potential host targets for broad-spectrum antiviral strategies. We will focus on the recent advances in Numb-associated kinases-related cellular functions and their potential as host targets for viral infections in this review. Questions that remained unknown on the cellular functions of Numb-associated kinases will also be discussed.

Introduction

The Numb-associated kinase family is ubiquitous in mammals, yeast, Drosophila, and other model organisms (Fig. 1A). It consists of diverse serine/threonine kinases sharing about 30% sequence identity within their kinase domains at N terminal but little conservation in other domains (Fig. 1B). In mammals, NAK family has four members: cyclin G-associated kinase (GAK), adaptor-associated kinase 1(AAK1), BMP-2-inducible kinase (BMP2K, also known as BIKE), and myristoylated and palmitoylated serine/threonine kinase 1 (MPSK1, also named STK16/TSF1/PKL12/Krct). Among them, AAK1 and BMP2K exhibit high homology in structure, while some functions of AAK1 and GAK are redundant [1]. In Saccharomyces cerevisiae, members of actin-regulating kinases (Ark family), such as Ark1, Prk1, and Akl1, belong to the Numb-associated kinase family. Ark1 and Prk1 exhibit high identity in their kinase domains, but almost no similarity in their non-kinase C terminal domains exists. In addition, the homology of the kinase domain between Saccharomyces cerevisiae Ark1 and human AAK1 is 38% [2]. Saccharomyces cerevisiae Env7 is an ortholog of human MPSK1 [3]. Drosophila melanogaster has two known homologs: Numb-associated kinase (NAK) and the clathrin-disassembly factor auxilin. Drosophila melanogaster NAK shares 33% sequence homology to Human MPSK1 across the kinase domain [1]. The ortholog of NAK in Caenorhabditis elegans is Sel-5. As Serine /threonine kinases, the Numb-associated kinases usually perform their functions by phosphorylating the serine and threonine residues of their substrates. Many phosphorylated substrates of Numb-associated kinases have been identified so far (Table 1).

Fig. 1.

(A) Phylogenetic tree of NAK family produced from alignments generated using MEGA7 software. C.e., Caenorhabditis elegans; D.m., Drosophila melanogaster; H.s., Homo sapiens; M.m., Mus musculus; S.c., Saccharomyces cerevisiae

(B) Domain organization of NAKs. The kinase domain is located at N-terminal. The length of the region downstream of the kinase domain is variable. DPF motif (yellow) is an α-adaptin–interacting motif. NPF motif (red) confers binding to EH domains. DLL motif (green) is known to mediate clathrin binding (CBD2). Only two proteins in the family have DNA J domains (purple), located at their C-terminus. Ark1 contains two conserved motifs PxPPPKP, and Prk1 contains one copy, which closes to the C-terminus of both proteins

Table 1.

Phosphorylated substrates of NAK family. Substrates and their phosphorylation sites reported for Numb-associated kinases are listed. Furthermore, the related physiological functions are also summarized in this table

Protein	Alias	Substrates	P-sites/motifs of the substrates	Physiological function	Reference
AAK1		AP-1	T154	Regulation of clathrin-dependent endocytosis; Endosomal pathway	[10] [8] [27]
		AP-2	T156
		Numb	T102	Dendritic morphogenesis of developing mammalian neurons
		Eps15	-	Positive regulation of Notch signaling pathway
GAK	Auxilin	AP-2	T156	Process clathrin coat disassembly; Receptor-mediated endocytosis	[55] [56] [57] [58]
		AP-1	-
		Atp1a3	T705	Na+/K+-ATPase
		CHC	T606	Mitosis; Microtubule generation and outgrowth
		Protein phosphatase 2 A	T104
BMP2K	BIKE	AP-2	T156	Clathrin-dependent endocytosis	[14] [59]
		CLINT1	T294	Cell signaling
MPSK1	Krct, EDPK, STK16, TSF-1, PKL12	MBP	-	Secretory vesicle trafficking	[60] [61] [62] [63]
		Histone H1	-
		PHAS-1	-	-
		DRG1	-	Cellular growth
PRK1	Prk1p	Pan1	The LxxQxTG motifs	Actin cortical patch assembly; Endocytosis; TORC2-Dependent Signaling Network in yeast	[64] [65] [66] [18] [67]
		Sla1	The LxxQxTG motifs
		Ent1/2	T394, T416
		Scd5	The LxxTxTG motifs
		Bni1	The LxxQxTG motifs	Exocytosis
ARK1	Ark1p	Pan1	The LxxQxTG motifs	Actin cortical patch assembly; Clathrin-dependent endocytosis; TORC2-Dependent Signaling Network in yeast	[64] [65] [18]
		Sla1	The LxxQxTG motifs
AKL1	Akl1p	Sal1	The LxxQxTG motifs	Clathrin-dependent endocytosis; TORC2-Dependent Signaling Network in yeast	[68] [22] [23]
		Pan1	-
		Ent1	-
		Fpk1	-
Nak		AP-2	T156	Endocytosis in Drosophila	[69] [70]
		Numb	-	Asymmetric cell division
Auxilin		AP-2	-	Clathrin coat disassembly; Receptor-mediated endocytosis; Compound eye morphogenesis; Notch signaling pathway	[55]
		AP-1	-

It is reported that the Numb-associated kinases are implicated in diverse cellular processes, including endocytosis, autophagy, dendrite morphogenesis, osteoblast differentiation, and the regulation of the Notch pathway. Therefore, they have been associated with a diversity of diseases and disorders such as neuropathic pain, Alzheimer’s disease, Parkinson’s disease, and prostate cancer. So far, members of the Numb-associated kinases have been considered as potential drug targets for related disorders including neuropathic pain and Parkinson’s disease [4, 5]. Recent researches indicate that Numb-associated kinases play a crucial role in viral replication. Human Numb-associated kinases are demonstrated to be implicated in the viral infection of HCV, DENV, EBOV, and rabies virus (RABV) [6]. COVID-19, caused by SARS-CoV-2, is still a threat to global health. Although vaccines can reduce the severity of COVID-19, vaccination for some special populations is limited, and the appearance of new viral variants is also a challenge. Thus, therapeutic antiviral drugs are needed. Silencing AAK1, BIKE, GAK, and MPSK1 individually with siRNA is reported to suppress SARS-CoV2 infection in Calu-3 cells. Moreover, inhibitors of Numb-associated kinases such as sunitinib, erlotinib, and gefitinib are demonstrated to suppress SARS-CoV-2 infection [7]. So Numb-associated kinases are also proposed as possible therapeutic targets for broad-spectrum antiviral strategies. We will focus on the recent advances in Numb-associated kinases-related cellular functions, and their potential as host targets for viral infections in this review.

Numb-associated kinases associated cellular functions

The role of Numb-associated kinases in endocytosis

Endocytosis is a fundamental cellular process that transports extracellular molecules, membrane proteins, and lipids into cells, and plays an essential role in nutrient uptake, receptor internalization, and signaling transduction. Depending on the cargo type and internalization mechanism, endocytosis has been divided into three major types: phagocytosis, pinocytosis, and receptor-mediated endocytosis. Numb-associated kinases play important roles in receptor-mediated endocytosis which includes clathrin-mediated endocytosis (CME), caveolae-mediated endocytosis, and clathrin and caveolae-independent endocytosis [10].

In mammals, AAK1 is a key regulator in receptor-mediated endocytosis. It has two physiological substrates: Numb and the plasma membrane adaptor complex AP2 (adaptor protein 2). On the one hand, AAK1 can phosphorylate Numb on Thr-102, thereby regulating the endocytic activity of Numb in clathrin-mediated endocytosis [8]. On the other hand, AAK1 is a binding partner for the medium subunits of the AP2 complex. AP2 acts as the bridge between cargo membrane proteins and clathrin lattice as a key component of the clathrin-mediated endocytic mechanism. Phosphorylation of the µ2 subunit of the AP2 complex on Thr156 is essential for the formation of coated vesicles during clathrin-mediated endocytosis. Earlier studies suggest that phosphorylation of µ2 subunit can increase the binding affinity between AP2 and membrane protein sorting signals [9]. AAK1 is a responsible kinase that phosphorylates the µ2 subunit of AP2 on Thr-156. The in vitro phosphorylation of µ2 subunit mediated by AAK1 is demonstrated to increase the binding affinity of AP2 to sorting signals [10]. Moreover, AAK1 localizes to the sites of endocytosis and copurifies with the AP-2 complex. All of this evidence strongly suggests that AAK1 is important in receptor-mediated endocytosis.

GAK, known as auxilin 2, is the ubiquitous homolog of the neuronal-specific protein auxilin 1, including an N-terminal kinase domain and a C terminal domain composed of the clathrin-binding domain, a J domain (DNA J domain), and a tensin domain [11]. GAK is reported to share some functions with AAK1. Similar to AAK1, GAK is also able to phosphorylate AP2 on Thr156 in vitro. But the kinase-dead mutant of GAK could rescue the phenotype caused by the knockdown of GAK in transferrin uptake assay, suggesting that the kinase activity of GAK is not essential to efficient endocytosis, or it is redundant by AAK1 [11]. Moreover, GAK is a cytosolic protein that is especially localized in the perinuclear area [12]. Its co-localization with AP1 and co-purification with the AP1 complex suggest that it may play an important role in the clathrin-dependent trafficking associated with the trans-Golgi network [11]. In addition, GAK binds to the heat shock cognate protein Hsc70 by its C terminus, playing an essential role in the uncoating of clathrin-coated vesicles as a cofactor to Hsc70 [12]. Both the clathrin-binding domain and the J domain in the C terminus of GAK are required for the clathrin uncoating activity of Hsc70. It is reported that the early stages of CME could be partially inhibited when GAK was silenced by specific siRNAs, and that GAK mutants without functional J domain cannot rescue this defect [11].

BMP2K is closely related to AAK1 in structure and also plays an important role in the regulation of clathrin-mediated endocytosis [13]. Shikha T. Ramesh et al. reported that BMP2K could regulate CME by phosphorylating AP2 on Thr-156 both in vivo and in vitro. They demonstrated that depletion of BMP2K obstructed AP2 phosphorylation resulting in defects in clathrin-coated pit morphology and internalization of cargo in Hela cells. BMP2K could bind with both α and β appendages of AP2 to be recruited to CCPs (clathrin-coated pits). However, it is the C terminus of BMP2K(561aa-1161aa) but not the kinase domain required for its interaction with AP2. BMP2K mutants without C terminus fail to interact with AP2 [14]. Moreover, BMP2K also interacts with the Numb and regulates the functions of the Numb in endocytosis, which is important for mammalian development [15].

Dynamic actin filaments are required for endocytosis. In Saccharomyces cerevisiae, endocytic vesicles have been demonstrated to move on the actin cable. Eps15-like protein Pan1 is essential both for actin cytoskeleton organization and endocytosis in yeast. On the one hand, Pan1 is involved in the activation of Arp2/3 which is the actin polymerization initiation complex. On the other hand, Pan1 could interact with multiple endocytic proteins and acts as a key scaffold protein in various events of receptor-mediated endocytosis. Studies show that Pan1 is a key regulator in the late stage of endocytosis in yeast [16]. Ark1/Prk1 kinases could regulate the interaction between actin cable and endocytic vesicles by phosphorylating coat proteins Pan1 and Ent1/2 [17, 18]. Phosphorylation of Pan1 by Ark1/Prk1 kinases leads to the disassembly of clathrin-coated vesicles. Sla1 is a key component of the endocytic machinery, which forms the Pan1-Sla1-End3 complex (yeast actin cytoskeleton regulatory complex) together with Pan1 and End3. Prk1 could regulate the dissociation of the Pan1-Sla1-End3 complex by phosphorylating Pan1 and Sla1. There is a functional overlap between Ark1 and Prk1. Depletion of ARK1 and PRK1 together results in severe abnormal actin cytoskeleton and endocytosis, whereas loss of neither Ark1 nor Prk1 causes significant defects [19, 20]. Although Ark1 and Prk1 share high identity in their kinase domain, there is almost no detectable similarity existing between the C terminal domains of Ark1 and Prk1. Studies show that the poly-P motif in the C terminus of Ark1 is responsible for its patch localization. A 21-aa motif in the C terminus of Prk1 is required for Prk1 to interact with Arp2 [19]. Similar to Prk1, Akl1 regulates endocytosis via phosphorylation of Pan1 in the Sla1-Pan1-End3 complex. Inhibition of the activity of Akl1 reduces the phosphorylation level of Pan1 and other endocytic coat proteins leading to slower endocytosis kinetics [21–23].

Modulation of Notch pathway

Numb-associated kinases are also involved in the regulation of the Notch pathway, which is relevant to various cellular processes such as cell proliferation, differentiation, and cell fate assignation [6, 24]. The Notch signaling pathway is normally activated by the interaction between the Notch receptor and the Notch ligand. Following engaging with ligands, the extracellular domain of the Notch receptor is cleaved by a disintegrin and metalloprotease. Subsequently, the intracellular domain is cleaved from the transmembrane domain by multiprotein γ-secretase complex and then translocated to the nucleus to regulate the transcription of target genes [25, 26].

AAK1 is demonstrated to directly interact with the activated metalloprotease-cleaved Notch to positively modulate the Notch signaling pathway. It is reported that overexpression of AAK1 contributes to the stabilization of activated Notch, while depletion of AAK1 results in reduced Notch transcriptional activity [27]. Several proteins implicated in CME have been demonstrated to participate in Notch trafficking in mammals, such as α-adaptin, Numb, Eps15, and clathrin [27, 28]. Numb, a substrate of Numb-associated kinases, regulates the Notch pathway negatively via binding to AP2 [29–31]. Numb is shown to inhibit monoubiquitination of the Notch intermediate and promote polyubiquitination of the intracellular domain of the Notch receptor to regulate its degradation. Thus, AAK1 and Numb act antagonistically in the notch pathway [27, 32]. Thereby AAK1 plays dual roles in the Notch pathway: the activation and redistribution of Numb and activation of the Notch.

The Notch pathway is evolutionarily conserved in all metazoans. Elimination of Sel-5 activity in C.elegans leads to inhibition of the constitutive activity of lin-12(d) which is similar to the metalloprotease-cleaved form of Notch [33]. In Drosophila, auxilin is required for the internalization of the Notch ligand Delta, which is critical for the activation of the Notch receptor [34]. The auxilin mutations in Drosophila interact specifically with Notch and disrupt several processes mediated by Notch [35].

The role of Numb-associated kinases in autophagy

Autophagy is a eukaryotic highly conserved degradation and recycling process in which cytoplasmic components including protein aggregates and damaged organelles are engulfed by autophagosomes and delivered to lysosomes or vacuoles for degradation [36]. Dysregulation of autophagy results in various diseases such as neurodegenerative diseases, cancer, and infections. Recent studies indicate that Numb-associated kinases are also involved in the regulation of autophagy. Jaroslaw Cendrowski et al. have reported that the two splicing variants of BMP2K played opposite roles in autophagy to modulate erythroid maturation. They found that BMP2K-L and BMP2K-S regulate SEC16A-dependent COPII assembly differentially by binding to SEC16A to modulate autophagy. BMP2K-L promoted autophagic degradation, whereas BMP2K-S inhibited autophagic degradation [37]. In addition, Autophagy machinery has been demonstrated to be essential to the internalization of MHC class I molecules. AAK1 seems to contribute to the internalization of MHC class I molecules depending on its recruitment by LC3B [38]. Mitophagy is a kind of selective autophagy to clear abnormal mitochondria, which is essential to maintain cellular homeostasis. Dysfunction of GAK is discovered to positively regulate PRKN-independent mitophagy which is essential in physiology and stress conditions [39].

Numb-associated kinases in dendrite morphogenesis, mitosis, cell differentiation

Other biological functions of Numb-associated kinases have also been reported in the relevant literature. AAK1 is shown to control dendrite development in hippocampal neurons as a substrate of NDR1 (Nuclear Dbf2-related kinase 1). Depletion of AAK1 with siRNA results in an increase of dendrite branching and length [40]. But how AAK1 contributes to limiting the growth of dendrites remains largely unknown. GAK can be implicated in mitotic progression by cooperating with clathrin. It is demonstrated that RNAi-mediated depletion of GAK causes activation of the spindle assembly checkpoint and multi-aster formation. The cell cycle will be arrested at metaphase in cells that lack GAK [41]. GAK is demonstrated to regulate microtubule outgrowth from kinetochores/chromatin via interaction with clathrin. Moreover, the absence of GAK results in a dramatical reduction of astral microtubules and defective spindle position during mitosis similar to the phenotype caused by the depletion of clathrin, indicating that GAK is essential for proper spindle positioning [42, 43]. The BMPs (bone morphogenic proteins) are reported to play a key role in skeletal development. Expression of BMP2K seems to be increased during osteoblast differentiation induced by BMP-2 in a mouse prechondroblastic cell line. When stably expressed, BMP2K negatively regulates osteoblast differentiation and mineral deposition. Thus, BMP2K, including a nuclear localization signal and a glutamine-rich region, is demonstrated to be implicated in the regulation of the cell differentiation process [44].

Numb-associated kinases as potential broad-spectrum antiviral targets

Numb-associated kinases are essential regulators of endocytosis which is a cellular process that could be hijacked by multiple viruses for their own benefit. Recent studies show that Numb-associated kinases participate in the life cycle of many viruses, thus they are proposed as potential host targets for the research and development of antiviral drugs. AAK1 is shown to be required for the infection of Rabies virus (RABV). AAK1 knockdown decreases, and AAK1 knockout inhibits, RABV infection in N2a cells [44]. Wang et al. reported that phosphorylation of AP2 by AAK1 was responsible for RABV entry. They also found that Sunitinib, an inhibitor of AAK1 and GAK, was able to increase the survival of mice challenged with RABV street virus [45]. Both AAK1 and GAK are reported to be implicated in the infection of HCV, DENV, and EBOV [46–48]. Gregory Neveu et al. have found that AAK1 and GAK could act on the assembly and entry of HCV in an AP2 phosphorylation-dependent manner [46, 47]. Moreover, Fei Xiao et al. demonstrated that AAK1 and GAK could regulate the cell-to-cell spread of HCV which is susceptibility to their inhibitors [49]. Furthermore, AAK1 and GAK are implicated in the entry and infectious virus production of multiple viruses such as DENV and EBOV depending on their binding partner APs. AAK1 or GAK inhibitors including sunitinib and erlotinib are demonstrated to have broad-spectrum antiviral potential to multiple RNA viruses such as HCV, DENV, and EBOV [48]. These data indicate that Numb-associated kinases are potential antiviral drug targets.

COVID-19 caused by SARS-CoV-2 still threatens global health. Recent studies have shown the involvement of Numb-associated kinases in the infection of SARS-CoV-2. Multiple inhibitors of AAK1 among FDA-approved drugs, including baricitinib, sunitinib, and erlotinib, are shown to block the infection of SARS-CoV-2 in the BenevolentAI’s knowledge graph [50], suggesting that AAK1 is a potential target for the treatment of COVID-19. In the absence of AAK1, BIKE, GAK, and MPSK1, the infection of SARS-CoV-2 in human lung epithelial cells is proved to be suppressed. In addition to erlotinib and sunitinib, 7-oxozeaenol which inhibits the activity of Numb-associated kinases also suppresses SARS-CoV-2 infection. Furthermore, Combination treatment with sunitinib and erlotinib exhibits a synergistic effect against SARS-CoV-2 infection [7]. In addition, inhibition of AAK1 by Jaktinib hydrochloride which is also a broad-spectrum JAK (Janus kinase) inhibitor is shown to slow the proliferation of SARS-CoV-2 [51]. The expression level of AAK1 in COVID-19 patients is significantly decreased compared with normal subjects, suggesting that COVID-19 patients may consume a large amount of AAK1 in the late stage of the disease [52]. All of the evidence strongly suggests that Pharmacological suppression of Numb-associated kinases can be a potential strategy to treat COVID-19. Identifying new therapeutic drugs against COVID-19 targeting Numb-associated kinases is on-going. Recently, Qin Lin et al. design and synthesize an analogue of baricitinib which has strong activity against AAK1, JAK1, and JAK2 compared to baricitinib. Baricitinib, an inhibitor of JAK1/2 is an approved drug that can regulate cytokine levels. Besides, Baricitinib also shows anti-viral activity by inhibiting AAK1 and GAK [53]. Thus, baricitinib has been used in the clinical investigation against SARS-CoV-2 infection [54].

Discussion

The Numb-associated kinase family widely exists in mammals, yeast, Drosophila, and other model organisms. They have various functions including endocytosis, autophagy, regulation of the Notch pathway, osteoblast differentiation, and dendrite morphogenesis. Since family members are now largely determined by the homology within kinase domains, it is important to better understand how kinase domains cooperate with the divergent C terminal of these proteins. Numerous studies on Numb-associated kinases have been performed in the last several decades. Members of this kinase family are discovered to participate in a diversity of cellular processes. But there are still many problems that need to be solved. AAK1 is highly homology to Saccharomyces cerevisiae Prk1, so it is speculated that Prk1 may also be involved in autophagy in Saccharomyces cerevisiae. Further experiments are needed to confirm this. GAK has been reported to be involved in dendrite morphogenesis, mitosis, and cell differentiation, but the details of how GAK works need to be further studied. In addition, there are still novel functions of these proteins that remain undetermined. Though Numb-associated kinases have been proposed as promising host targets for the treatment of COVID-19, it is challenging to design and synthesize inhibitors with high specificity. Numb-associated kinases play essential roles in clathrin-mediated endocytosis. It is not known if the disruption of endocytosis will result in undesirable side effects. More work is needed to understand their cellular functions and related regulation mechanisms to have a more comprehensive understanding of those kinases.

Abbreviations

AAK1

adaptor-associated kinase 1

AP1

adaptor protein 1

AP2

adaptor protein 2

BMP2K

BMP-2-inducible kinase

CCPs

Clathrin-coated pits

CME

Clathrin-mediated endocytosis

COVID-19

Coronavirus disease 2019

DENV

dengue virus

EBOV

Ebola virus

GAK

cyclin G-associated kinase

HCV

hepatitis C virus

Hsc70

heat shock cognate protein

JAK

Janus kinase

MPSK1

myristoylated and palmitoylated serine/threonine kinase 1

NAK

Numb-associated kinase

NDR1

Nuclear Dbf2-related kinase 1

RABV

Rabies virus

SARS-CoV-2

severe acute respiratory syndrome coronavirus 2

siRNA

Small interfering RNA

Funding

The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.

Data Availability

Data sharing not applicable to this article as no datasets were generated or analysed during the current study.

Declarations

Ethics approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Competing Interests

The authors declare no conflict of interest.