FAM150A and FAM150B are activating ligands for Anaplastic Lymphoma Kinase

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*For correspondence: ruth.
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Received: 01 July 2015
Accepted: 28 September 2015
Published: 29 September 2015
Reviewing editor: Roger Davis,
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FAM150A and FAM150B are activating
ligands for anaplastic lymphoma kinase
Jikui Guan
1†
, Ganesh Umapathy
1†
, Yasuo Yamazaki
1
, Georg Wolfstetter
1
,
Patricia Mendoza
1
, Kathrin Pfeifer
1
, Ateequrrahman Mohammed
1
,
Fredrik Hugosson
1
, Hongbing Zhang
2
, Amy W Hsu
2
, Robert Halenbeck
2
,
Bengt Hallberg
1
, Ruth H Palmer
1
*
1
Department of Medical Biochemistry and Cell Biology, Instititute of Biomedicine,
Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden;
2
Five Prime
Therapeutics Inc., South San Francisco, United States
Abstract Aberrant activation of anaplastic lymphoma kinase (ALK) has been described in a
range of human cancers, including non-small cell lung cancer and neuroblastoma (Hallberg and
Palmer, 2013). Vertebrate ALK has been considered to be an orphan receptor and the identity of
the ALK ligand(s) is a critical issue. Here we show that FAM150A and FAM150B are potent ligands
for human ALK that bind to the extracellular domain of ALK and in addition to activation of wild-
type ALK are able to drive ’superactivation’ of activated ALK mutants from neuroblastoma. In
conclusion, our data show that ALK is robustly activated by the FAM150A/B ligands and provide an
opportunity to develop ALK-targeted therapies in situations where ALK is overexpressed/activated
or mutated in the context of the full length receptor.
DOI: 10.7554/eLife.09811.001
Introduction
Activation of anaplastic lymphoma kinase (ALK) is commonly due to fusion of the ALK kinase
domain with a dimerization partner that drives activation, however, ALK activation also occurs in the
context of the full length receptor, for example, as activating point mutations in
neuroblastoma (Maris et al., 2007;Care
´n et al., 2008;Chen et al., 2008;George et al., 2008;
Janoueix-Lerosey et al., 2008;Mossee
´et al., 2008;Hallberg and Palmer, 2013). In many addi-
tional tumor types ALK overexpression and activation has been described, and it is unclear whether
this is dependent on the activity of a ligand (Hallberg and Palmer 2013). The ALK receptors in both
Drosophila and Caenorhabditis elegans have well defined ligands – Jeb (Englund et al., 2003;
Lee et al., 2003;Stute et al., 2004) and HEN-1 (Ishihara et al., 2002), respectively. In contrast, ver-
tebrate ALK has long been considered as an orphan receptor.
The human ALK locus encodes a classical receptor tyrosine kinase (RTK) comprising a unique
extracellular ligand-binding domain, a transmembrane domain and an intracellular tyrosine kinase
domain (Hallberg and Palmer, 2013). The extracellular portion of ALK which contains two MAM
domains (named after meprin, A-5 protein and receptor protein tyrosine phosphatase m), a glycine-
rich region (GR) and a LDLa domain, is unique among the RTKs. ALK, and the related leukocyte tyro-
sine kinase (LTK) RTK, share kinase domain similarities as well as a GR in the membrane proximal
portion of their extracellular domains (ECDs) (Iwahara et al., 1997;Morris et al., 1997). Recent
screening of the extracellular proteome identified two novel secreted proteins as ligands for LTK –
family with sequence similarity 150A (FAM150A) and family with sequence similarity 150B
(FAM150B). Both bind to the ECD of the receptor leading to activation of downstream signaling in
cell culture models (Zhang et al., 2014). FAM150A and FAM150B are unique, displaying homology
only with one another but not with any other proteins in mammals (Zhang et al., 2014).
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Furthermore, we found the reported strong expression of FAM150B in the human adrenal gland
(Zhang et al., 2014) intriguing, given the role of ALK in neuroblastoma.
Here we report the identification of FAM150A and FAM150B as potent ligands for human ALK.
We investigated ALK activation by FAM150A and FAM150B proteins in PC12 cell neurite outgrowth
assays where we observed a strong activation of ALK signaling. Conditioned medium containing
either FAM150A or FAM150B was able to activate endogenous ALK signaling in neuroblastoma
cells. We also employed the model organism Drosophila melanogaster as a readout for activation of
ALK by FAM150A and FAM150B, showing that FAM150 proteins are able to robustly drive human
ALK activation when ectopically coexpressed in the fly. FAM150A and FAM150B bind to the ECD of
ALK and, in addition to activation of wild-type ALK, are able to drive ‘superactivation’ of activated
ALK mutants from neuroblastoma. The GR of the ALK receptor ECD is important for FAM150 activa-
tion, and monoclonal antibodies (mAb) recognizing the GR of ALK are able to inhibit activation of
ALK by FAM150A. In conclusion, our data show that ALK is robustly activated by FAM150A/B finally
providing an answer to the identity of the elusive ligands for this RTK.
Results and discussion
ALK and the related LTK share similarity in their membrane proximal ECD in the form of a glycine-
rich domain that is ~250 amino acids in length (Figure 1A, GR depicted in grey). This domain con-
tains multiple runs of up to eight glycine residues, and is unique to ALK and LTK within the human
genome. The importance of the GR in ALK has been highlighted in Drosophila studies, where four
independent point mutations leading to exchange of single glycine residues result in complete loss
of function in vivo (Englund et al., 2003) (Figure 1—figure supplement 1). The similarity between
ALK and LTK within the GR is ~70%, with amino acid identity of 55%, containing a total of 51 con-
served glycine residues (Figure 1B). Given this similarity, and the important role of the glycine-rich
domain for function in Drosophila, we hypothesized that FAM150A and FAM150B, which were
recently reported as ligands for LTK (Zhang et al., 2014) may act as ligands for ALK.
eLife digest Cells have receptor proteins on their surface that enable them to detect changes in
their environment and communicate with other cells. Signal molecules bind to a segment of the
receptor called the extracellular domain that faces out from the cell. This can result in the activation
of another domain in the receptor that is just inside the cell, which, in turn, activates signaling
pathways that relay the information around the cell.
However, these communication systems are often disrupted in cancer cells. This helps the cells to
override the strict growth controls imposed upon them by other (healthy) cells in the body. The
gene that encodes a receptor protein called Anaplastic Lymphoma Kinase (or ALK for short) is often
mutated in some types of human cancer so that the protein is always active. However, we still do not
know what signal molecules bind to the ALK protein to activate it in normal cells.
Guan, Umapathy et al. used a variety of cell biology and biochemical techniques to study the role
of ALK. The experiments show that when either of two proteins called FAM150A and FAM150B are
produced in rat nerve cells alongside ALK, the nerve cells rapidly respond and form outgrowths.
Experiments using cancer cells derived from human nerve cells also yielded similar results. Guan,
Umapathy et al. found that the extracellular domain of ALK can physically interact with FAM150A
and FAM150B.
The eyes of fruit flies that had been genetically modified to produce the human ALK protein
alongside either FAM150A or FAM150B grew more than normal, giving the eyes an abnormal
"rough" appearance. Further experiments showed that FAM150A and FAM150B are also able to
increase the level of activation of an ALK mutant protein that is already active. Therefore, in future,
the development of drugs that stop FAM150A and FAM150B from binding to ALK may be useful for
treating cancers that are driven by high levels of ALK activity. Many challenging questions lie ahead
to better understand how FAM150A and FAM150B interact with ALK.
DOI: 10.7554/eLife.09811.002
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Figure 1. FAM150A and FAM150B activate ALK. (A) Schematic overview of human anaplastic lymphoma kinase
(ALK) and leukocyte tyrosine kinase (LTK) protein domain structures. ALK and LTK share a membrane proximal
extracellular glycine-rich region (GR, grey), transmembrane and an intracellular tyrosine kinase domain (red). In
addition, the extracellular region of ALK contains two MAM domains (purple) and an LDLa-motif (yellow). (B)
Alignments of the GR of ALK and LTK, conserved runs of glycine residues are highlighted in bold with red
asterisks. (C) Neurite outgrowth in PC12 cells expressing either vector control, FAM150A, FAM150B, ALK,
FAM150A and ALK, FAM150B and ALK or ALK-F1174L quantified in (D). Experiments were performed in triplicate
and each sample within an experiment was performed in duplicate (error bars indicate SD). (E) Whole cell lysates
from PC12 cells expressing either vector control, FAM150A, ALK, FAM150A and ALK or ALK-F1174L were analyzed
by immunoblot analysis of ALK, pALK-Y1604 (arrowheads), FAM150A and ERK1/2. Pan-ERK was employed for
equal loading. (F) Whole cell lysates from PC12 cells expressing either vector control, FAM150B, ALK, FAM150B
Figure 1. continued on next page
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In order to initially test whether FAM150A or FAM150B could activate ALK we assayed neurite
outgrowth activity in PC12 cells. Expression of either FAM150A, or FAM150B or ALK alone, did not
lead to activation. This was in contrast to the strong activation seen with the positive control ALK-
F1174L which is a well characterized constitutively active ALK neuroblastoma mutation. However,
coexpression with either FAM150A or FAM150B led to robust activation of ALK neurite outgrowth
activity (Figure 1C, quantified in 1D). This ALK activation was visualized by phosphorylation of
Y1604 in the tail of the ALK intracellular domain (Figure 1E, F;Figure 1—figure supplement 2) and
was further associated with stimulation of downstream signaling such as the phosphorylation of the
downstream target ERK1/2 (Figure 1E, F). Thus in PC12 cells FAM150A and FAM150B act to stimu-
late signaling via the wild-type ALK receptor, in a manner that leads to phosphorylation of the ALK
receptor.
We next tested the ability of exogenously produced FAM150A or FAM150B to activate ALK,
employing medium conditioned with FAM150A or FAM150B to activate PC12 cells expressing ALK.
Addition of conditioned medium from cells expressing FAM150A or FAM150B led to strong activa-
tion of ALK in receiving cells that expressed ALK, as measured by neurite outgrowth and activation
of downstream signaling (Figure 2A, B). We then examined whether ALK signaling activity induced
by either FAM150A or FAM150B was sensitive to ALK inhibition, employing the ALK inhibitor
crizotinib (Zou et al., 2007). We observed that ALK activation by FAM150A or FAM150B was inhib-
ited by addition of 250 nM crizotinib (Figure 2A, B).
To address whether endogenous levels of ALK responded to medium conditioned with either
FAM150A or FAM150B, we employed the IMR-32 neuroblastoma cell line that expresses wild-type
ALK. Once again, we observed robust activation of ALK by the addition of either FAM150A-contain-
ing or FAM150B-containing conditioned medium, employing pALK-Y1604 and pERK1/2 as readout
(Figure 2C). The activation of ALK and downstream signaling events in IMR-32 cells were effectively
blocked by the addition of 250 nM crizotinib (Figure 2C). The activation of exogenous ALK in PC12
cells or endogenous ALK in IMR-32 cells was reproduced with the addition of recombinant
FAM150A purified from Sf21 cells (Figure 2D). Unfortunately we could not produce sufficient
amounts of recombinant FAM150B protein, precluding a similar analysis with FAM150B.
More careful examination of ALK activation by FAM150A or FAM150B containing conditioned
media in IMR-32 cells revealed activation of signaling at 2–5 min, peaking around 20 min before
decreasing at later time points. This response was observed at the level of ALK receptor phosphory-
lation (pALK-Y1604), and with downstream pERK5 (Umapathy et al., 2014) and pERK1/2
(Figure 2E, F). Here we employed antibodies recognizing pALK-Y1604, allowing differentiation of
ALK activation in IMR-32 cells from that of LTK (Figure 1—figure supplement 2). Thus we observed
that exogenously produced FAM150A and FAM150B proteins stimulate ALK signaling in both PC12
cells and IMR-32 cells. We also examined potential modulation of ALK activation by FAM150A/B
upon addition of heparin, which has recently been reported to activate ALK (Murray et al., 2015).
While we were able to see strong binding of purified FAM150A protein as well as FAM150A-HA and
FAM150B-HA from conditioned medium to heparin-agarose, we did not observe any additional acti-
vation of ALK by FAM150 proteins in the presence of heparin (Figure 2—figure supplement 1; Fig-
ure 2—figure supplement 2).
We next analyzed the ability of FAM150A or FAM150B to activate human ALK in a Drosophila
model, which offers a clear readout. Neither the Drosophila Alk ligand Jeb (Englund et al., 2003;
Lee et al., 2003;Stute et al., 2004) nor previously proposed vertebrate ligands, that is, human mid-
kine (MDK) and pleiotrophin (PTN) are able to activate either mouse or human ALK (Yang et al.,
Figure 1. Continued
and ALK or ALK-F1174L were analyzed by immunoblot analysis of ALK, pALK-Y1604 (arrowheads), HA (FAM150B)
and pERK1/2. Pan-ERK was employed for equal loading.
DOI: 10.7554/eLife.09811.003
The following figure supplements are available for Figure 1:
Figure supplement 1. Glycine residues in the glycine-rich region of Drosophila ALK are critical for function.
DOI: 10.7554/eLife.09811.004
Figure supplement 2. Conservation of phosphoepitopes in the intracellelular domains of ALK and LTK.
DOI: 10.7554/eLife.09811.005
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Figure 2. Conditioned medium containing either FAM150A or FAM150B activates endogenous ALK. (A) Neurite
outgrowth in PC12 cells expressing either vector control or anaplastic lymphoma kinase (ALK) were cultured in
medium from Human Embryonic Kidney (HEK) 293 cells transfected with either FAM150A or FAM150B, quantified
below. Experiments were performed in triplicate and each sample within an experiment was performed in
duplicate. Values represent mean ±SD from at least three independent experiments. (B) Whole cell lysates from
PC12 cells expressing either vector control or ALK stimulated with medium from HEK293 cells transfected with
vector control, FAM150A or FAM150B were analyzed by immunoblot. Analysis was carried out in the presence or
absence of 250 nM crizotinib. Detection of ALK activation was visualized with pALK-Y1604 (arrowheads), ALK and
pERK1/2 in whole cell lysates. The presence of FAM150A in supernatants was confirmed with anti-FAM150A
antibodies, while the presence of FAM150B-HA was confirmed with anti-HA antibodies. Pan-ERK was employed
for equal loading. (C) IMR32 cells harboring a wild-type ALK receptor were stimulated for 20 min with medium
from HEK293 cells transfected with either vector control, FAM150A or FAM150B prior to analysis by immunoblot.
Figure 2. continued on next page
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2007;Hugosson et al., 2014). Expression of either FAM150A or FAM150B in the developing
eye, using the GMR-Gal4 driver, resulted in normal eye morphology (Figure 3A). In contrast, expres-
sion of constitutively active ALK-F1174S described in neuroblastoma patients results in a rough eye
morphology (Figure 3A) (Martinsson et al., 2011), while no eye phenotype was observed upon the
expression of wild-type human ALK alone (Figure 3A) (Martinsson et al., 2011,Schonherr, Ruuth
et al., 2011,Schonherr, Ruuth et al., 2011,Chand et al., 2013;Hugosson et al., 2014). This can
be compared with coexpression of either FAM150A or FAM150B together with human ALK which
led to a rough eye phenotype, proving that both FAM150A and FAM150B were able to activate
human ALK in this in vivo system (Figure 3A, B).
We next examined binding of the human ALK ECD with purified FAM150A by ELISA and Biacore
surface plasmon resonance (SPR) analysis. Both ELISA and Biacore analysis showed that FAM150A
binds specifically to the ALK ECD (Figure 4A;Figure 4—figure supplement 1). Purified human
FAM150A (Zhang et al., 2014) binds to ALK-ECD-Fc with a dissociation constant of ~20 nM
(Figure 4A). We also examined the ability of either FAM150A or FAM150B to bind human ALK by
immunoprecipitation and immunofluorescence analysis (Figure 4B–D). Here we observed that both
FAM150A and FAM150B could be independently immunoprecipitated with the ALK receptor, and
that ALK could be immunoprecipitated with either FAM150A or FAM150B in the reciprocal pull-
down experiments (Figure 4B, C;Figure 4—figure supplement 2). Since FAM150A/B also bind
LTK, we asked whether ALK and LTK interacted in the presence of FAM150A. Indeed, both ALK and
FAM150A were found to immunoprecipitate with LTK (Figure 4—figure supplement 3). Immunoflu-
orescence analysis of cells expressing human ALK confirmed these findings, with clear association of
both HA-tagged FAM150A and HA-tagged FAM150B with ALK expressing cells observed
(Figure 4D). In these experiments we observed uptake of FAM150A and FAM150B into ALK-positive
intracellular vesicles, suggesting that binding to ALK on the cell surface leads to uptake and internal-
ization of the ALK-FAM150 complex (Figure 4D).
We further examined the role of the GR of ALK in FAM150A/B binding. Deletion of the GR in the
ALK ECD did not affect FAM150A binding but led to loss of FAM150B binding (Figure 4–—figure
supplement 4). Given the importance of the GR of ALK in Drosophila (Englund et al., 2003) (Fig-
ure 1–—figure supplement 1), we mutated these conserved residues in human ALK (G740D,
G823D, G893E and G934D) and tested their effect on the FAM150-ALK interaction. Despite being
well expressed, all four glycine mutations in the GR severely impaired binding of FAM150B to ALK.
Consistent with our earlier results we were unable to see any effect of mutation of individual glycine
on the FAM150A-ALK interaction (Figure 4—figure supplement 5).
In previous work, we and others have generated antibodies to the ECD of ALK (Moog-Lutz et al.,
2005,Witek et al., 2015). We investigated whether any of these antibodies recognized the GR in
the ALK ECD, and were able to identify three interesting candidates (anti-ALK mAb13, mAb48 and
Figure 2. Continued
Analysis was carried out in the presence or absence of 250 nM crizotinib. Stimulation with the ALK activating
antibody mAb46 was employed as positive control. Detection of ALK activation was visualized with ALK, pALK-
Y1604 (arrowheads) and pERK1/2. Pan-ERK was employed for equal loading. (D) IMR32 cells harboring a wild-type
ALK receptor stimulated with increased amounts of recombinant His-tagged FAM150A purified from Sf21 cells.
Detection of ALK activation was visualized with ALK, pALK-Y1278 (arrowheads) and pERK1/2. Pan-ERK was
employed for equal loading. (E) Time course of IMR32 cells stimulated with FAM150A conditioned medium.
Stimulation with ALK activating antibody mAb46 was employed as positive control. Detection of ALK activation
was visualized with ALK, pALK-Y1604 (arrowheads), pERK5 and pERK1/2. Pan-ERK was employed for equal
loading. (F) Time course of IMR32 cells harboring a wild type ALK receptor stimulated with FAM150B conditioned
medium. Stimulation with ALK activating antibody mAb46 was employed as positive control. Detection of ALK
activation was visualized with ALK, pALK-Y1604 (arrowheads), pERK5 and pERK1/2. Pan-ERK was employed for
equal loading.
DOI: 10.7554/eLife.09811.006
The following figure supplements are available for Figure 2:
Figure supplement 1. FAM150 proteins interact with heparin.
DOI: 10.7554/eLife.09811.007
Figure supplement 2. Investigation of the effect of Heparin and FAM150A on ALK activation.
DOI: 10.7554/eLife.09811.008
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Figure 3. Expression of either FAM150A or FAM150B is sufficient for the activation of wild-type ALK. (A) Ectopic
expression of either FAM150A or FAM150B together with the wild-type human anaplastic lymphoma kinase (ALK)
receptor in the Drosophila eye with the GMR-Gal4 driver disrupts the highly organized pattern of ommatidia of the
Drosophila eye and generates a rough eye phenotype. Images of adult Drosophila eyes ectopically expressing
Figure 3. continued on next page
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mAb135, Figure 4—figure supplement 6). These antibodies were next tested for their ability to
modulate the activation of ALK by purified FAM150A. While mAb135 had no effect on FAM150A
activation of ALK, we observed that mAb13 exhibited strong inhibition of FAM150A-induced ALK
activation (Figure 4E), while mAb48 robustly activated ALK in the absence of FAM150A ligand
(Figure 4E). Taken together, these data strongly support a role for the GR in the activation of ALK
by FAM150 proteins.
In cell lines as well as in the Drosophila model, we observed extremely high levels of ALK activa-
tion when compared even with highly active ALK mutants such as ALK-F1174S, suggesting the possi-
bility that these already active mutants can be activated to a higher level in the presence of a potent
ligand. Therefore, we examined the ability of either FAM150A or FAM150B to ‘superactivate’
already active ALK mutants as described in neuroblastoma. In PC12 cells we indeed observed activa-
tion of the ALK-R1275Q mutant to a higher level, as evidenced by increased pERK1/2 and in neurite
outgrowth assays (Figure 4F). A similar effect was observed with the ALK-F1174L mutant (Figure 4—
figure supplement 7). This increased activity was also seen in CLB-GAR cells, which endogenously
express the ALK-R1275Q mutant, with increased activation of ERK1/2 upon the addition of either
FAM150A or FAM150B (Figure 4G).
In conclusion, our work identifies FAM150A and FAM150B as novel ligands that bind and activate
the human ALK RTK. The identity of a ligand(s) for vertebrate ALK has been an evolving question for
many years, with a number of reports over the past 15 years examining the role of the small heparin
binding growth factors MDK and PTN as ligands for vertebrate ALK (Stoica et al., 2001;
Stoica et al., 2002). However, a number of independent studies including our own work have pre-
sented convincing evidence that MDK and PTN do not activate ALK (Motegi et al., 2004;Moog-
Lutz et al., 2005;Mourali et al., 2006;Mathivet et al., 2007;Hugosson et al., 2014;
Murray et al., 2015). The recent identification of long chain heparins as activators of ALK in neuro-
blastoma cells (Murray et al., 2015) and the interplay of this with ALK activation by FAM150A and
FAM150B will require further work. FAM150A and FAM150B are very basic proteins; FAM150A has
a predicted pI of 10.6, while FAM150B has one of 9.75, and we show here that FAM150A exhibits
heparin binding properties, however we have not observed a cooperative role of heparin and the
FAM150 ligands in ALK activation as has been described for the fibroblast growth factor receptors
and FGF. Our data also suggest that FAM150 ligands can bind an ALK-LTK heterodimer complex.
While the physiological significance of such an interaction is currently unclear, ALK and LTK have
been reported to be coexpressed in some tissues, such as the mouse hippocampus (Weiss et al.,
2012), where such an interaction may be important.
FAM150 proteins do not seem to be conserved outside the vertebrates. In zebrafish there are
three FAM150 proteins (FAM150ba, FAM150bb and FAM150A), while in mouse and humans only
FAM150A and FAM150B exist (Zhang et al., 2014). In invertebrate model organisms the ALK
ligands, Jeb in Drosophila and HEN-1 in C. elegans, do not appear to resemble the FAM150A/B
ligands. The Drosophila Jeb ligand is unable to activate either mouse or human ALK (Yang et al.,
2007), and to date, no Jeb-like ligand for vertebrate ALKs has been reported. The evolution of the
FAM150 proteins as ALK ligands is an interesting topic for further investigation.
In the case of ALK mutations that are ligand dependent, as well as in situations where the ALK
receptor is overexpressed, such as in neuroblastoma, the mechanisms by which ALK is activated is
fundamental for understanding their role in tumor development. Here the identification of ALK
ligands will allow a more rigorous interrogation of ALK signaling in these scenarios. The finding
that FAM150A and FAM150B are not only able to activate the wild-type receptor, but also to ‘super-
activate’ mutant ALK receptors, is important in this context. These findings, together with the
observed expression pattern of FAM150A and FAM150B in adrenal gland and thyroid and the
Figure 3. Continued
wild-type ALK in the presence of either FAM150A or FAM150B are shown. Controls expressing either wild-type
ALK, FAM150A or FAM150B alone do not display a rough eye phenotype. The constitutively active ALK-F1174S
mutant was employed as positive control. (B) Phenotypes observed in adult flies upon GMR-Gal4 driven
expression of either FAM150A or FAM150B together with the wild-type ALK at two different temperatures, 18˚C
and 25˚C.
DOI: 10.7554/eLife.09811.009
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Figure 4. FAM150A and FAM150B bind to ALK and further activate signaling mediated by the R1275Q ALK
neuroblastoma mutation. (A) Binding kinetics of purified FAM150A to extracellular domain of anaplastic lymphoma
kinase (ALK-ECD-Fc) in a Biacore surface plasmon resonance (SPR) analysis. (B) FAM150A immunoprecipitates with
human ALK. Immunoprecipitation with either anti-FLAG(DYKDDDDK)(ALK) or anti-HA (FAM150A) was performed
and the resulting immunoprecipitates immunoblotted for the presence of ALK (blue arrowheads) and
FAM150A (red arrowheads), *indicates immunoglobulin light and heavy chains. (C) FAM150B immunoprecipitates
with human ALK. Immunoprecipitation with either anti-FLAG (ALK) or anti-HA (FAM150B) was performed and the
resulting immunoprecipitates immunoblotted for the presence of ALK (blue arrowheads) and FAM150B (red
arrowheads),*indicates immunoglobulin light and heavy chains. (D) Human Embryonic Kidney (HEK) 293 cells
expressing ALK were incubated with either control or HA-tagged FAM150A or HA-tagged FAM150B conditioned
Figure 4. continued on next page
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inhibition of ALK activation by FAM150A with monoclonal antibodies shown here, suggest that a
future potential therapeutic arm may involve agents that interfere with FAM150A/B activation of
ALK offering additional therapeutic approaches in patients with neuroblastoma for which single drug
treatments targeting ALK have had limited effect to date.
Materials and methods
Antibodies and inhibitors
The primary antibodies used were anti-pan-ERK (1:10,000; BD Transduction Laboratories), anti-ALK
(for immunofluorescence 1:1000; ab4061, Abcam), anti-ALK (D5F3, 1:5000; Cell Signaling Technol-
ogy), anti-ALK mAb135 (1:2000; [Witek et al., 2015]), anti-pERK5 (1:1000; Cell Signaling Technol-
ogy), anti-pALK-Y1278 (1:2000; Cell Signaling Technology), anti-pALK-Y1604 (1:2000; Cell Signaling
Technology) and anti-pERK1/2-T202/Y204 (1:2000; Cell Signaling Technology), anti-FAM150A
(1:4000, Atlas Antibodies), anti-HA (1:1000 for immunofluorescence, 1:6000 for immunoblotting;
16B12, Covance). The activating monoclonal antibody mAb46 and ALK monoclonal antibodies
mAb13, mAb48 were a kind gift from M. Vigny and have been described previously (Moog-
Lutz et al., 2005). The ALK inhibitor crizotinib was purchased from Chem Express (Shanghai).
Figure 4. Continued
medium prior to analysis by immunohistochemistry. Both HA-tagged FAM150A and HA-tagged FAM150B bind to
ALK-expressing cells. Higher magnification panels indicate intracellular vesicles positive for both ALK and HA-
tagged FAM150A/B. (E) NB1 and IMR32 neuroblastoma cells were treated with 2 mg/ml monoclonal antibodies
(mAB13, mAb48 or mAb135) prior to stimulation with FAM150A. Detection of ALK activation was visualized with
pALK-Y1604 (arrowheads) and pERK1/2 in whole cell lysates. Pan-ERK or tubulin were employed for equal loading.
(F) Whole cell lysates from PC12 cells expressing either vector control or ALK-R1275Q were stimulated with
medium from HEK293 cells transfected with vector control, FAM150A or FAM150B prior to analysis by
immunoblot. Analysis was carried out in the presence or absence of 250 nM crizotinib. Detection of ALK activation
was visualized with pALK-Y1604 (arrowheads) and pERK1/2 in whole cell lysates. Pan-ERK was employed for equal
loading. Neurite outgrowth was performed in triplicate and each sample within an experiment was performed in
duplicate (error bars indicate SD). (G) CLB-GAR cells harboring the ALK-R1275Q mutant were stimulated for 30
min with medium from HEK293 cells transfected with either vector control, FAM150A or FAM150B prior to analysis
by immunoblot. Analysis was carried out in the presence or absence of 250 nM crizotinib. Detection of ALK
signaling activation was visualized with pERK1/2. Pan-ERK was employed for equal loading. pERK1/2 intensity was
analyzed from three independent experiments (error bars indicate SD).
DOI: 10.7554/eLife.09811.010
The following figure supplements are available for Figure 4:
Figure supplement 1. FAM150A binds to the extracellular domain of human ALK by ELISA.
DOI: 10.7554/eLife.09811.011
Figure supplement 2. ALK interacts with both FAM150A and FAM150B.
DOI: 10.7554/eLife.09811.012
Figure supplement 3. ALK and LTK interact in the presence of FAM150A. FAM150A and anaplastic lymphoma
kinase (ALK) coimmunoprecipitate with leukocyte tyrosine kinase (LTK).
DOI: 10.7554/eLife.09811.013
Figure supplement 4. Effect of deletion of the glycine rich domain of ALK on FAM150A and FAM150B binding
DOI: 10.7554/eLife.09811.014
Figure supplement 5. Effect of glycine mutations in the glycine-rich domain of ALK on FAM150A and FAM150B
binding.
DOI: 10.7554/eLife.09811.015
Figure supplement 6. Identification of monoclonal antibodies recognising the glycine-rich region of the ALK
ECD.
DOI: 10.7554/eLife.09811.016
Figure supplement 7. FAM150A and FAM150B bind to ALK and further activate signaling mediated by the ALK-
F1174L neuroblastoma mutant.
DOI: 10.7554/eLife.09811.017
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Short report Cell biology

Immunofluorescence
Human embryonic kidney (HEK) 293 cells were grown on collagen-coated cover slips in 24-well
plates, prior to Lipofectamine transfection with either pcDNA3-ALK or pcDNA3 alone as control.
Conditioned medium containing FAM150A-HA or FAM150B-HA was then incubated for 20 min prior
to fixation and immunostaining. For immunostaining of HEK cells, cells were fixed with 4% parafor-
maldehyde/Dulbecco’s Modified Eagle Medium (DMEM) and blocked with 50 mM NH
4
Cl/
phosphate buffered saline (PBS). After permeabilization with 0.3% Triton X-100 and 5% goat serum
containing PBS, cells were incubated with primary antibody as indicated overnight at 4˚C. For visuali-
zation cells were further incubated with fluorescence-labeled secondary antibody followed by analy-
sis on LSM710 or LSM800 confocal microscopes (Zeiss).
Cell lysis and immunoblotting
PC12 cells cotransfected with wild-type ALK, together with either FAM150A or FAM150B, were incu-
bated for 24 hr prior to starvation in serum-free DMEM for a further 24 hr. PC12 cells expressing
human wild-type ALK were serum starved for 24 hr prior to stimulation with FAM150A or FAM150B
conditioned medium for 30 min. IMR-32 cells were stimulated with either 0.85 mg/ml mAb46 as posi-
tive control (Moog-Lutz et al., 2005), or with FAM150A or FAM150B conditioned medium for 20
min unless otherwise stated. For ALK inhibition, crizotinib was employed at a final concentration of
250 nM. Both PC12 and IMR-32 cells were lysed in 1 sodium dodecyl sulfate (SDS) sample buffer,
and precleared lysates were run on SDS-PAGE, followed by immunoblotting using the indicated anti-
bodies. ALK downstream activation was detected by anti-pERK1/2. Pan-ERK was used as loading
control. ALK phosphorylation was checked by using pALK-Y1278 and pALK-Y1604 antibodies. Cell
lysis and immunoblotting was performed according to protocols described
previously (Schonherr, Yang et al., 2010).
Immunoprecipitation
To detect the interaction between ALK and FAM150A or FAM150B, HEK293 cells (in 10 cm dishes,
80–90% confluent) were transfected with 5 mg of pcDNA3-ALK-FLAG together with either 5 mg of
pTT5-FAM150A-HA or pcDNA3-FAM150B-HA. As controls, cells were transfected with 5 mg of
pcDNA3 vector together with 5 mg of pcDNA3-ALK-FLAG or pTT5-FAM150A-HA or pcDNA3-
FAM150B-HA, correspondingly. Cells were lysed 16–20 hr after transfection, in 1 ml of lysis buffer
(50 mM Tris-Cl, pH7.4, 250 mM NaCl, 1% Triton X-100 and proteinase inhibitor cocktail) on ice for
10 min prior to clarification by centrifugation at 14,000 rpm at 4º˚C for 15 min. Supernatant (30 ml)
was taken and used as input control, and the remaining sample was incubated with either 20 ml of
anti-FLAG M2 magnetic beads (Sigma) or 20 ml of anti-HA agarose beads (ThermoScientific) corre-
spondingly for 2 hr at 4º˚C. The beads were then washed five times with lysis buffer and boiled in 80
ml of 1 x SDS sample buffer. Both inputs and immunoprecipitation products were separated by 9%
SDS-PAGE gel. ALK and FAM150A or ALK and FAM150B were detected in the same blot with anti-
ALK mAb135 (1:2000) and anti-HA antibody (1:6000, Covance), respectively.
Neurite outgrowth assay
PC12 (2 x 10
6
) cells were co-transfected with either 0.3 mg of empty pcDNA3 vector control,
pcDNA3-ALK or pcDNA3-ALK-F1174L together with 0.5 mg pEGFPN1 (Clontech) and either 0.5 mg
pTT5-FAM150A-HA or pcDNA3-FAM150B-HA as indicated by electroporation using Amaxa electro-
porator (Amaxa Biosystems). Cells were resuspended in 100 ml Ingenio electroporation solution
(Mirus Bio LCC) prior to transfection. After transfection, cells were kept
in minimum essential medium (MEM) supplemented with 7% horse serum and 3% fetal bovine serum
and seeded into 24-well plates. After 24 hr of incubation, the fraction of Green fluorescent
protein (GFP)-positive and neurite carrying cells versus GFP-positive cells was observed under a Zeiss
Axiovert 40 CFL microscope. PC12 cells incubated with FAM150A-conditioned or FAM150B-condi-
tioned media were analyzed for neurite outgrowth 24 hr after addition of conditioned media. For
ALK inhibition, crizotinib was employed at a final concentration of 250 nM. To be judged as a neurite
carrying cell, the neurite of the cell should be at least twice the diameter of a normal cell body.
Experiments were performed in triplicates, and each sample within an experiment was performed in
duplicate.
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Short report Cell biology

Generation of FAM150A-conditioned and FAM150B-conditioned media
HEK293 cells, at ~90% confluency in 10 cm dishes, were transfected with 8.0 mg of either pcDNA3
vector control, pTT5-FAM150A-HA or pcDNA3-FAM150B-HA with Lipofectamine 2000 (Invitrogen).
After 6 hr, medium was replaced with non-supplemented MEM and the subsequent conditioned
medium was harvested 48 hr post transfection. For IMR-32 stimulation experiments 1 ml of either
control, FAM150A-conditioned or FAM150B-conditioned medium was added to IMR-32 cells seeded
in 6-well plates resulting in a total final volume of 3 ml. For PC12 stimulation experiments, medium
was replaced with either control, FAM150A-conditioned or FAM150B-conditioned medium as
indicated.
Generation and purification of FAM150A-His
DNA encoding FAM150A (amino acids 21–129) was cloned into pFastBac-HBM TOPO (Invitrogen)
and used to transform competent DH10Bac cells (Invitrogen) generating the recombinant FAM150A
bacmid. Sf21 cells were transformed with the bacmid DNA using Cellfectin II reagent (Invitrogen) in
serum-free Grace’s Medium (Invitrogen). Supernatant from Sf21 cells containing recombinant
FAM150A virus was collected after 72 hr and used for virus amplification. For protein expression,
Sf21 cells were grown to a cell density of 1–1.5 10
6
cells/ml in Sf900-II medium (Gibco) containing
1% fetal bovine serum, penicillin and streptomycin, and infected with FAM150A recombinant viral
stock. Medium was harvested 72 hr after infection and protein was affinity-purified on Ni-NTA aga-
rose resin (Qiagen).
Monoclonal antibody modulation of ALK activation by FAM150A
Wild-type ALK expressing neuroblastoma cell lines NB1 and IMR-32 were treated with either 2 mg/
ml of FAM150A, 2 mg/ml of one of the following monoclonal antibodies: mAb13, mAb48, and
mAb135, or combinations of FAM150A and either of the three antibodies respectively for 20 min. In
case of the combination of FAM150A and antibodies, antibodies were added to the cells 30 min
prior to addition of FAM150A. The activation of ALK by FAM150A was visualized with pALK-Y1604
and pERK1/2. Total ALK and tubulin were employed as internal controls.
Expression of human ALK mutants in the Drosophila eye
The Gal4-UAS expression system was used for the ectopic expression of human ALK in Drosophila
eye (Brand and Perrimon 1993). cDNAs encoding either FAM150A or FAM150B were cloned into
pUAST and verified by sequencing. Transgenic flies carrying pUAST-FAM150A or pUAST-FAM150B
were generated by BestGene, Inc., and crossed with pGMR-Gal4, UAS-ALK-WT (Martinsson et al.,
2011), where hALK expression was driven by pGMR-Gal4 (Bloomington Stock Center) in the devel-
oping eye. UAS-ALK-F1174S (Martinsson et al., 2011) driven by pGMR-Gal4 was included as a posi-
tive control. Scale bar indicates 100 mm. Images were taken on a Zeiss AxioZoomV16
stereomicroscope.
Binding of FAM150A to ALK-ECD-Fc by ELISA
FAM150A was purified as described (Zhang et al., 2014). For ELISA, 384-well white Maxisorp (Nunc
Nalge) were coated overnight at 4º˚C with 20 ml of 2 mg/ml FAM150A diluted in 100 mM sodium
bicarbonate buffer, pH 9.6. Subsequent steps were performed at room temperature. Plates were
washed with PBST (PBS, 0.05% Tween20) and blocked for 1 hr with blocking/dilution buffer (PBST
with 1% bovine serum albumin [BSA]). Plates were washed and 20 ml/well of ALK-Fc, LTK-Fc, and
control-Fc diluted in blocking buffer added and incubated for 2 hr. Plates were washed and subse-
quently 20 ml/well of 1:10,000 dilution of horseradish peroxidase conjugated anti-human IgG (Jack-
son Labs) was added and incubated for 1 hr. After washing, 20 ml/well of ELISA Pico
Chemiluminescent Substrate (Thermo Fisher Pierce) was added and incubated for 5 min before read-
ing on a plate reader.
Binding of FAM150A to ALK-ECD-Fc by Biacore analysis
Binding kinetics of FAM150A to LTK-ECD-Fc and ALK-ECD-Fc fusion proteins was determined using
Biacore T100 SPR (GE Healthcare Life Sciences, Piscataway, NJ). LTK-ECD-Fc and ALK-ECD-Fc were
captured on a CM4 sensor chip immobilized with anti-human IgG antibody using the human
Guan et al. eLife 2015;4:e09811. DOI: 10.7554/eLife.09811 12 of 16
Short report Cell biology

antibody capture kit (GE Healthcare Life Sciences, Piscataway, NJ). 4-(2-hydroxyethyl)-1-piperazinee-
thanesulfonic acid (HEPES) buffered saline10 mM, pH 7.4 with 0.05% Tween20 20 mg/ml heparin
sodium salt (HBS-P, GE Healthcare Life Sciences, Piscataway, NJ, Sigma Aldrich, St Louis, MO) was
used as the running and dilution buffer. Capture levels of the ECD-Fc fusions were adjusted to
~300–500 resonance units (RU). FAM150A was injected at eight concentrations (200, 66.6, 22.2, 7.4,
2.46, 0.82, 0.27 nM, and a 0 nM control) for 120 s and dissociation was followed for 180 s. The asso-
ciation constant, dissociation constant, and affinity for FAM150A for LTK and ALK Fc-ECD fusions
were calculated using the Biacore T100 evaluation software package using standard double
referencing technique and the 1:1 binding model.
Materials and methods for figure supplements
Heparin binding of purified FAM150A-His and FAM150A/B from
conditioned media
Purified FAM150A-His (25 mg) in 20 mM Tris-HCl in a total volume of 1 ml was incubated with 20 ml
of heparin-agarose (H0402, Sigma) overnight at 4º˚C followed by 5 washing with 20 mM Tris HCl,
50 mM NaCl. Bound proteins were eluted by boiling in 70 ml 20 mM Tris-HCl, 0.9 M NaCl with load-
ing buffer and eluates were separated on 15% SDS-PAGE prior to immunoblot with anti-FAM150A
antibody. Purified FAM150A-His 5 mg was employed as input control. Conditioned media from cells
transfected with pcDNA3 control or pTT5-FAM150A-HA or pcDNA3-FAM150B-HA was incubated
with 30 ml of heparin-agarose (H0402, Sigma) for 3 hr at 4º˚C followed by 5 washing with lysis
buffer. Bound proteins were eluted by boiling in 50 ml 4SDS sample buffer and eluates were sepa-
rated on 13% SDS-PAGE prior to immunoblot with anti-HA antibody. Conditioned media 50 ml was
employed as input control.
Stimulation of ALK by FAM150A in the presence of heparin
IMR-32 cells were treated with either 1 mg/ml (final concentration) of purified FAM150A 753 alone or
a combination of 1 mg/ml of FAM150A and 10 mg/ml of heparin (H3149, Sigma) 754 for the indicated
time points (5, 10, 20, 30 and 60 min). Cell lysates were subjected to immunoblotting with anti-
pERK1/2 antibody to visualize the activation effect. Tubulin was employed as loading control. NB1
cells were treated with heparin alone at the indicated concentrations (1, 10 and 100 mg/ml), 2 mg/ml
of FAM150A alone, or a combination of 10 mg/ml of heparin and 2 mg/ml of FAM150A for 10 min
prior to immunoblotting analysis. pALK (Y1604) and pERK1/2 antibodies were used to evaluate the
activation effect. Total ALK and panERK were used as loading controls.
Coimmunoprecipitation and immunoblotting
HEK293 cells (10 cm dishes, 80–90% confluent) were transfected with 5 mg of pcDNA3-ALK 5 mg of
pTT5-FAM150A-HA, or 5 mg of pcDNA3-ALK 5 mg of pcDNA3-FAM150B-HA, respectively. Around
20 hr after transfection, cells were lysed, clarified and equally divided prior to incubation with either
2mg of mouse IgG control (Santa Cruz), 2 mg of ALK mAb31, or 2 mg of mouse anti-HA (Covance)
antibody. After overnight incubation at 4º˚C, cell lysates with antibodies were further incubated with
15 ml of protein-G sepharose (GE healthcare) for another 2 hr. After 4 washes with lysis buffer,
agarose beads were boiled in 2 SDS sample buffer. Eluates, as well as 1/50th of cell lysate (input),
were separated on 9% SDS-PAGE. ALK and FAM150A/B were detected in the same blot employing
anti-ALK mAb135 and anti-HA antibodies.
To investigate interaction between ALK, LTK and FAM150A, HEK293 cells in 10 cm dishes were
transfected with 3 mg of pcDNA3-LTK-HA 4 mg of pTT5-FAM150A, or 3 mg of pcDNA3-LTK-HA 3 mg
of pcDNA3-ALK 4 mg of pTT5-FAM150A, respectively. Around 20 hr after transfection, cells were
lysed, clarified and equally divided prior to incubation with 30 ml of mouse IgG agarose (A0919,
Sigma), or 15 ml of mouse anti-HA agarose (ThermoScientific), respectively, for 2 hr at 4˚C. After 4 
washes, agarose beads were boiled in 2 SDS sample buffer. Eluates, as well as 1/50th of cell lysate
(input), were separated in 9% SDS-PAGE. ALK, LTK and FAM150A were detected in the same blot
employing anti-ALK mAb135, anti-HA and anti-FAM150A antibodies.
To test whether the glycine-rich region (GR) of the ALK extracellular domain (ECD) is involved in
binding of FAM150A/B, a GR-deleted version of ALK (ALK-delGR) was generated by overlap exten-
sion PCR to remove the entire GR of ALK (amino acids 689–940). In addition, ALK with mutations in
Guan et al. eLife 2015;4:e09811. DOI: 10.7554/eLife.09811 13 of 16
Short report Cell biology

the conserved glycines G740D, G823D, G893E and G934D were also generated to test whether
mutation of these conserved glycines affects the binding of FAM150A/B. For coimmunoprecipitation
with ALK-delGR, HEK293 cells in 10 cm dishes were transfected with 4 mg of pcDNA3-ALK 4 mg of
pTT5-FAM150A-HA, or 4 mg of pcDNA3-ALK-delGR 4 mg of pTT5-FAM150A-HA, or 4 mg of
pcDNA3-ALK 4 mg of pcDNA3-FAM150B-HA, or 4 mg of pcDNA3-ALK-delGR 4 mg of pcDNA3-
FAM150B-HA, respectively. For coimmunoprecipitation with different ALK glycine mutants, 4 mg of
pTT5-FAM150A-HA or 4 mg of pcDNA3-FAM150B-HA were cotransfected with 4 mg of pcDNA3-
ALK (WT) or ALK bearing different glycine mutations (G740D, G823D, G893E and G934D) into
HEK293 cells in 10 cm dishes, respectively. The same coimmunoprecipitation procedure was fol-
lowed as described above for ALK, LTK and FAM150A. ALK and FAM150A/B were detected in the
same blot employing anti-ALK (D5F3) and anti-HA antibodies.
Characterization of monoclonal antibodies recognizing the ALK ECD
HEK293 cells transfected with control vector, ALK and ALK-delGR were lysed 24 hr after transfec-
tion. Cell lysates were subjected to immunoblotting analysis with different ALK monoclonal antibod-
ies (mAb13, mAb48 and mAb135 recognizing the extracellular portion of ALK; D5F3 recognizing the
intracellular portion of ALK).
Acknowledgements
The authors acknowledge the important contribution to this study, particularly in the spirit of free
exchange of scientific reagents and knowledge, made by Lewis T. Williams who generously provided
reagents and facilitated collaboration in this study. We thank the Centre for Cellular Imaging (CCI) at
the University of Gothenburg for providing confocal imaging support and Anne Uv for critical read-
ing of the manuscript. This work was supported by grants from the Swedish Cancer Society (BH 12-
0722, RHP 12-0796), the Children’s Cancer Foundation (BH 14/150, RHP 13/0049), the Swedish
Research Council (RHP 621-2011-5181, BH 521-2012-2831), and the SSF Programme Grant (RB13-
0204).
Additional information
Funding
Funder Grant reference number Author
Cancerfonden (Swedish
Cancer Society)
12-0796 Ruth H Palmer
Vetenskapsra˚ det 621-2011-5181 Ruth H Palmer
Barncancerfonden (Swedish
Childhood Cancer Foundation)
13/0049 Ruth H Palmer
Stiftelsen fo
¨r Strategisk
Forskning
13-0204 Bengt Hallberg
Ruth H Palmer
Cancerfonden (Swedish
Cancer Society)
12-0722 Bengt Hallberg
Vetenskapsra˚ det 521-2012-2831 Bengt Hallberg
Barncancerfonden (Swedish
Childhood Cancer Foundation)
14/150 Bengt Hallberg
The funders had no role in study design, data collection and interpretation, or the decision to
submit the work for publication.
Author contributions
JG, GU, YY, GW, PM, KP, FH, HZ, AWH, RH, BH, RHP, Conception and design, Acquisition of data,
Analysis and interpretation of data, Drafting or revising the article; AM, Acquisition of data, Analysis
and interpretation of data
Author ORCIDs
Ruth H Palmer, http://orcid.org/0000-0002-2735-8470
Guan et al. eLife 2015;4:e09811. DOI: 10.7554/eLife.09811 14 of 16
Short report Cell biology

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