Histidine phosphorylation-mediated signal transduction regulates axon regeneration in C. elegans

Afshar K, Willard FS, Colombno K, Johnston CA, McCudden CR, Siderovski

DP, Gönczy P (2004) RIC-8 is required for GPR-1/2-dependent Ga function

during asymmetric division of C. elegans embryos. Cell 119: 219–230

Attwood PV, Piggott MJ, Zu XL, Besant PG (2007) Focus on phosphohistidine.

Amino Acids 32: 145–156

Brenner S (1974) The genetics of Caenorhabditis elegans. Genetics 77: 71–94

Busam RD, Thorsell A-G, Flores A, Hammarström M, Persson C, Hallberg BM

(2006) First structure of a eukaryotic phosphohistidine phosphatase. J Biol

Chem 281: 33830–33834

Cai X, Srivastava S, Surindran S, Li Z, Skolnik EY (2014) Regulation of the

2+

epithelial Ca

channel TRPV5 by reversible histidine phosphorylation

mediated by NDPK-B and PHPT1. Mol Biol Cell 25: 1244–1250

Chen L, Wang Z, Ghosh-Roy A, Hubert T, Yan D, O'Rourke S, Bowerman B, Jin

Y, Chisholm AD (2011) Axon regeneration pathways identified by

systematic genetic screening in C. elegans. Neuron 71: 1043–1057

Ch'ng Q, Williams L, Lie YS, Sym M, Whangbo J, Kenyon C (2003)

Identification of genes that regulate a left-right asymmetric neuronal

migration in Caenorhabditis elegans. Genetics 164: 1355–1367

Consortium C (2015) Sparse whole-genome sequencing identifies two loci for

major depressive disorder. Nature 523: 588–591

Crawley O, Opperman KJ, Desbois M, Adrados I, Borgen MA, Giles AC,

Duckett DR, Grill B (2019) Autophagy is inhibited by ubiquitin ligase activity

in the nervous system. Nat Commun 10: 5017

Cuello F, Schulze R, Heemeyer F, Meyer HE, Lutz S, Jakobs KH, Nicroomand

F, Wieland T (2003) Activation of heterotrimeric G proteins by a high energy

31

phosphate transfer via nucleoside diphosphate kinase (NDPK) B and Gb

subunits. J Biol Chem 278: 7220–7226

Di L, Srivastava S, Zhdanova O, Sun Y, Li Z, Skolnik EY (2010) Nucleoside

diphosphate kinase B knock-out mice have impaired activation of the K

channel Kca3.1, resulting in defective T cell activation. J Biol Chem 285:

38765–38771

Dokshin GA, Ghanta KS, Piscopo KM, Mello CC (2018) Robust genome editing

with short single-stranded and long, partially single-stranded DNA donors in

Caenorhabditis elegans. Genetics 210: 781–787

Ek P, Petterson G, Ek B, Gong F, Li J-P, Zetterqvist O (2002) Identification and

characterization of a mammalian 14-kDa phosphohistidine phosphatase: a

mammalian 14-kDa phosphohistidine phosphatase. Eur J Biochem 269:

5016–5023

Fuhs SR, Hunter T (2017) pHisphorylation: the emergence of histidine

phosphorylation as a reversible regulatory modification. Curr Opin Cell Biol

45: 8–16

Fuhs SR, Meisenhelder J, Aslanian A, Ma L, Zagorska A, Stankova M, Binnie A,

AI-Obeidi F, Mauger J, LemKe G et al (2015) Monoclonal 1- and 3phosphohistidine antibodies: new tools to study histidine phosphorylation.

Cell 162: 198–210

Gotta M, Ahringer J (2001) Distinct roles for Gb and Gbg in regulating spindle

position and orientation in Caenorhabditis elegans embryos. Nat Cell Biol 3:

297–300

Hardman G, Perkins S, Brownridge PJ, Clarke CJ, Byrne DP, Campbell AE,

Kalyuzhnyy A, Myall A, Eyers PA, Jones AR et al (2019) Strong anion

exchange-mediated phosphoproteomics reveals extensive human noncanonical phosphorylation. EMBO J 38: e100847

32

Hartsough MT, Morrison DK, Salerno M, Palmieri D, Ouatas T, Mair M, Patrick

J, Steeg PS (2002) Nm23-H1 metastasis suppressor phosphorylation of

kinase suppressor of Ras via a histidine protein kinase pathway. J Biol

Chem 277: 32389–32399

Hess HA, Roper JC, Grill SW, Koelle MR (2004) RGS-7 completes a receptorindependent heterotrimeric G protein cycle to asymmetrically regulate

mitotic spindle positioning in C. elegans. Cell 119: 209–218

Hess JF, Bourret RB, Simon MI (1988) Histidine phosphorylation and

phosphoryl group transfer in bacterial chemotaxis. Nature 336: 139–143

Hindupur SK, Colombi M, Fuhs SR, Matter MS, Guri Y, Adam K, Cornu M,

Piscuoglio S, Ng CKY, Betz C et al (2018) The protein histidine

phosphatase LHPP is a tumour suppressor. Nature 555: 678–682

Hippe H-J, Luedde M, Lutz S, Koehler H, Eschenhagen T, Frey N, Katus H,

Wieland T, Niroomand F (2007) Regulation of cardiac cAMP synthesis and

contractility by nucleoside diphosphate kinase B/G protein bg dimer

complexes. Circ Res 100: 1191–1199

Hippe H-J, Lutz S, Cuello F, Knorr K, Vogt A, Jakobs KH, Wieland T,

Niroomand F (2003) Activation of heterotrimeric G proteins by a high energy

phosphate transfer via nucleoside diphosphate kinase (NDPK) B and Gb

subunits. J Biol Chem 278: 7227–7233

Hippe H-J, Wolf NM, Abu-Taha I, Mehringer R, Just S, Lutz S, Niroomand F,

Postel EH, Katus HA, Rottbauer W et al (2009) The interaction of

nucleoside diphosphate kinase B with Gbg dimers controls heterotrimeric G

protein function. Proc Natl Acad Sci USA 106: 16269–16274

Hisamoto N, Sakai Y, Ohta K, Shimizu T, Li C, Hanafusa H, Matsumoto K

(2021) CDK14 promotes axon regeneration by regulating the non-canonical

Wnt signaling pathway in a kinase-independent manner. J Neurosci

41:8309–8320.

33

Huang X, Cheng HJ, Tessier-Lavigne M, Jin Y (2002) MAX-1, a novel

PH/MyTH4/FERM domain cytoplasmic protein implicated in netrin-mediated

axon repulsion. Neuron 34: 563–576

Hurley JH, Young LN (2017) Mechanisms of autophagy initiation. Annu Rev

Biochem 86: 225–244

Jansen G, Weinkove D, Plasterk RH (2002) The G protein g-subunit gpc-1 of

the nematode C. elegans is involved in taste adaptation. EMBO J 21: 986–

994

Kalagiri R, Adam K, Hunter T (2020) Empirical evidence of cellular histidine

phosphorylation by immunoblotting using pHis mAbs. Methods Mol Biol

2077: 181–191

Klumpp S, Hermesmeier J, Selke D, Baumeister R, Kellner R, Krieglstein J

(2002) Protein histidine phosphatase: a novel enzyme with potency for

neuronal signaling. J Cereb Blood Flow Metab 22: 1420–1424

Klumpp S, Krieglstein J (2009) Reversible phosphorylation of histidine residues

in proteins from vertebrates. Sci Signal 2: pe13

Ko S-H, Apple EC, Liu Z, Chen L (2020) Age-dependent autophagy induction

after injury promotes axon regeneration by limiting NOTCH. Autophagy 16:

2052–2068

Lai T, Garriga G (2004) The conserved kinase UNC-51 acts with VAB-8 and

UNC-14 to regulate axon outgrowth in C. elegans. Development 131: 5991–

6000

Lecroisey A, Lascu I, Bominaar A, Veron M, Delepierre M (1995)

Phosphorylation mechanism of nucleoside diphosphate kinase: 31P-nuclear

magnetic resonance studies. Biochemistry 34: 12445–12450

34

Li C, Hisamoto N, Nix P, Kanao S, Mizuno T. Bastiani M, Matsumoto K (2012)

The growth factor SVH-1 regulates axon regeneration in C. elegans via the

JNK MAPK cascade. Nat Neurosci 15: 551–557

Lu Z, Hunter T (2018) Metabolic kinases moonlighting as protein kinases.

Trends Biochem Sci 43: 301–310

Mahoney TR, Luo S, Nonet ML (2006) Analysis of synaptic transmission in

Caenorhabditis elegans using an aldicarb-sensitivity assay. Nat Protoc 1:

1772–1777

Masoudi N, Fancsalszky L, Pourkarimi E, Vellai T, Alexa A, Reményi A, Gartner

A, Mehta A, Takács-Vellai K (2013) The NM23-H1/H2 homolog NDK-1 is

required for full activation of Ras signaling in C. elegans. Development 140:

3486–3495

Mello CC, Kramer JM, Stinchcombm D, Ambros V (1991) Efficient gene transfer

in C. elegans: extrachromosomal maintenance and integration of

transforming sequences. EMBO J. 10: 3959–3970

Nix P, Hisamoto N, Matsumoto K, Bastiani M (2011) Axon regeneration requires

coordinate activation of p38 and JNK MAPK pathways. Proc Natl Acad Sci

USA 108:10738–10743.

Ogura K, Goshima Y (2006) The autophagy-related kinase UNC-51 and its

binding partner UNC-14 regulate the subcellular localization of the Netrin

receptor UNC-5 in Caenorhabditis elegans. Development 133: 3441–3450

Oldham WM, Hamm HE (2008) Heterotrimeric G protein activation by Gprotein-coupled receptors. Nat Rev Mol Cell Biol 9: 60–71

Panda S, Srivastava S, Li Z, Vaeth M, Fuhs SR, Hunter T, Skolnik EY (2016)

Identification of PGAM5 as a mammalian protein histidine phosphatase that

plays a central role to negatively regulate CD4 T cells. Mol Cell 63: 457–

469

35

Pastuhov SI, Fujiki K, Nix P, Kanao S, Bastiani M, Matsumoto K, Hisamoto N

(2012) Endocannabinoid–Goa signalling inhibits axon regeneration in

Caenorhabditis elegans by antagonizing Gqa–PKC–JNK signalling. Nat

Commun 3: 1136

Robatzek M, Niacaris T, Steger K, Avery L, Thomas JH (2001) eat-11 encodes

GPB-2, a Gb5 ortholog that interacts with Goa and Gqa to regulate C.

elegans behavior. Curr Biol 11: 288–293

Sakai Y, Hanafusa H, Pastuhov SI, Shimizu T, Li C, Hisamoto N, Matsumoto K

(2019) TDP2 negatively regulates axon regeneration by inducing

SUMOylation of an Ets transcription factor. EMBO Rep 20:e47517.

Sakai Y, Hanafusa H, Shimizu T, Pastuhov SI, Hisamoto N, Matsumoto K

(2021a) BRCA1–BARD1 regulates axon regeneration in concert with the

Gqa–DAG signaling network. J Neurosci 41:2842–2853.

Sakai Y, Tsunekawa M, Ohta K, Shimizu T, Pastuhov SI, Hanafusa H,

Hisamoto N, Matsumoto K (2021b) The Integrin signaling network promotes

axon regeneration via the Src–Ephexin–RhoA GTPase signaling axis. J

Neurosci 41:4754–4767.

Sakamoto R, Byrd DT, Brown HM, Hisamoto N, Matsumoto K, Jin Y (2005) The

Caenorhabditis elegans UNC-14 RUN domain protein binds to the kinesin-1

and UNC-16 complex and regulates synaptic vesicle localization. Mol Biol

Cell 16: 483–496

Shimizu T, Pastuhov SI, Hanafusa H, Matsumoto K, Hisamoto N (2018) The C.

elegans BRCA2-ALP/Enigma complex regulates axon regeneration via a

Rho GTPase-ROCK-MLC phosphorylation pathway. Cell Rep 24:1880–

1889.

Srivastava S, Li Z, Ko K, Choudury P, Albaqumi M, Johnson AK, Yan Y, Backer

JM, Unutmaz D, Coetzee WA et al (2006) Histidine phosphorylation of the

potassium channel KCa3.1 by nucleoside diphosphate kinase B is required

for activation of KCa3.1 and CD4 T cells. Mol. Cell 24: 665–675

36

Srivastava S, Li Z, Soomro I, Sun Y, Wang J, Bao L, Coetzee WA, Stanley CA,

Li C, Skolnik EY (2018) Regulation of KATP channel trafficking in

pancreatic b-cells by protein histidine phosphorylation. Diabetes 67: 849–

860

Srivastava S, Panda S, Li Z, Fuhs SR, Hunter T, Thiele DJ, Hubbrd SR, Skolnik

EY (2016) Histidine phosphorylation relieves copper inhibition in the

mammalian potassium channel KCa3.1. eLife 5: e16093

Srivastava S, Zhdanova O, Skolnik EY (2008) Protein histidine phosphatase 1

negatively regulates CD4 T cells by inhibiting the K channel KCa3.1. Proc

Natl Acad Sci USA 105: 14442–14446

Swanson RV, Alex LA, Simon MI (1994) Histidine and aspartate

phosphorylation: two-component systems and the limits of homology.

Trends Biochem Sci 19: 485–490

van der Voorn L, Gebbink M, Plasterk A, Ploegh HL (1990) Characterization of

a G-protein b-subunit gene from the nematode Caenorhabditis elegans. J

Mol Biol 213: 17–26

Wang B, Kundu M (2017) Canonical and noncanonical functions of ULK/Atg1.

Curr Opin Cell Biol 45: 47–54

Wieland T, Hippe HJ, Ludwig K, Zhou XB, Korth M, Klumpp S (2010) Reversible

histidine phosphorylation in mammalian cells: a teeter-totter formed by

nucleoside diphosphate kinase and protein histidine phosphatase 1.

Methods Enzymol 471: 379–402

Yan D, Wu Z, Chisholm AD, Jin Y (2009) The DLK-1 kinase promotes mRNA

stability and local translation in C. elegans synapses and axon

regeneration. Cell 138: 1005–1018

37

7. 図および表

His-kinase

NDPK

His

His

Protein

Protein

PHPT1

pHis-phosphatase

NDPK

(Human)

NDP Kinase

115

115

NDK-1

(C. elegans)

PHPT1

(Human)

152 aa

N I I H G S D 121

N I C H G S D 121 68% identity

NDP Kinase

153 aa

pHis PPase

52

44

PHIP-1

(C. elegans)

Y H A D 55

F H D D 47

125 aa

41% identity

pHis PPase

116 aa

km96 (frameshift insertion)

LHPP

(Human)

HAD hydrolase

270 aa

42% identity

LHPP-1

(C. elegans)

HAD hydrolase

km97 (frameshift deletion)

266 aa

38

Figure 1.

His-kinase and pHis-phosphatase.

Protein His-phosphorylation is regulated by His-kinase and pHisphosphatase.

NDK-1, PHIP-1, and LHPP-1 structures. Schematic diagrams of NDK-1,

PHIP-1, LHPP-1, and their mammalian counterparts are shown. The NDP

kinase domain is shown in green, and the phosphohistidine (pHis)

phosphatase domain in blue. Kinase-dead NDK-1(H118N) and

catalytically inactive PHIP-1(H45A) mutations are denoted by asterisks.

Identical and similar residues are highlighted with black and gray shading,

respectively. Arrowheads indicate premature stop codons caused by km96

and km97 mutations.

39

D-type motor neurons

Relative axon length = X/Y

phip-1(km96)

WT

Punc-25::ndk-1

ns

NS

ns

NS

✱✱✱

✱✱✱

Relative axon length

1.0

0.8

0.6

0.4

0.2

0.0

(n)

WT

phip-1

(km96)

(46)

(45)

phip-1

phip-1

(km96)

(km96)

Punc-25:: Punc-25::

phip-1 phip-1(H45A)

(49)

(65)

lhpp-1

(km97)

(63)

ns

NS

ns

NS

✱✱

Relative axon length

1.0

0.8

0.6

0.4

0.2

0.0

(60)WT

(37)

(n)

(45)

Punc(54) 25::

(46)

Puncphip-1

(60) 25::

ndk-1 ndk-1(H118N) (km96)

(44)

(41)

(53)

(56)

phip-1

(km96)

Punc-25::

ndk-1

(50)

(64)

40

Figure 2.

Protein His-phosphorylation inhibits axon regeneration.

Scheme for axotomy and relative axon length measurements of

GABAergic D-type motor neurons in C. elegans. Relative axon length was

determined by the distance from the ventral nerve cord to the injured axon

tip (X) normalized by the distance from the ventral nerve cord to the dorsal

nerve cord (Y).

Representative D-type motor neurons in wild-type animals, phip-1(km96)

mutants, and NDK-1-overexpressing animals 24 h after laser surgery.

Arrowheads indicate the tip of axotomized axons. Scale bar, 10 µm.

C, D Relative axon length 24 h after laser surgery at the young adult stage. The

number (n) of axons examined from three biological replicates is indicated.

The black bar in each violin plot indicates the median. **P < 0.01, ***P <

0.001, as determined by the Kruskal-Wallis test and Dunn’s multiple

comparison test. NS, not significant.

41

DBD

PHIP-1

(H45A)

PHIP-1

(H45A)

vector

AD

Growth

vector

GPB-1

GPB-1

266

(Human)

GNB1

Gβ ELMTYSHDNII

Human

GNB1

(C. elegans)

GPB-1

C. elegans

GPB-1 ELAMYSHDNII

(C. elegans)

GPB-2

C. elegans

GPB-2 QVCVYEKESIL

ns

ns

ns

ns

NS

✱✱✱✱

✱✱✱

0.6

0.4

0.2

Relative axon length

0.8

0.8

0.6

0.4

0.2

0.0

0.0

(n)

1.0

1.0

Relative axon length

Relative axon length

1.0

ns

✱✱

✱✱

✱✱✱

0.8

0.6

0.4

0.2

0.0

WT

gpb-1

(H266F)

WT

phip-1

(km96)

(49)

(38)

(56)

phip-1

phip-1

phip-1

lhpp-1

WT25:: Punc25:: Punc25::

phip-1

Puncgpb-1

(km96)

(km96)

(km96)

(km97)

ndk-1

ndk-1

(km96);

ndk-1

(H266F)

(H118N)

++

gpb-1

Punc-25:: PuncPunc-25

25::::

(H266F)

phip-1 phip-1(H45A)

ndk-1

(47)

(41)

(51)

phip-1

(km96)

phip-1

(km96)

Punc-25:

ndk-1

42

Figure 3.

NDK-1 and PHIP-1 regulate axon regeneration through His-

phosphorylation of the Gb subunit GPB-1.

PHIP-1 interaction with GPB-1 by yeast two-hybrid assay. The reporter

strain PJ69-4A was co-transformed with expression vectors encoding

GAL4 DBD-PHIP-1(H45A) and GAL4 AD-GPB-1, as indicated. Yeast

strains carrying the indicated plasmids were cultured on a selective plate

lacking histidine and containing 5 mM 5-aminotriazole for 4 days.

His-phosphorylation site in Gb. Sequence alignments of the Hisphosphorylation site and flanking amino acids among human GNB1, GPB1, and GPB-2 are shown. Identical and similar residues are highlighted

with black and gray shading, respectively. The His-phosphorylation site,

His-266, is indicated by an arrowhead.

Relative axon length 24 h after laser surgery at the young adult stage. The

number (n) of axons examined from three biological replicates is indicated.

The black bar in each violin plot indicates the median. **P < 0.01, ***P <

0.001, as determined by the Kruskal-Wallis test and Dunn’s multiple

comparison test. NS, not significant.

43

phip-1(km96)

3XFLAG::gpb-1

Heat (95℃)

WT

H266F

T7-GPC-2

HA-GPB-1

GST-NDK-1

(kDa)

100

75

IB:

50

WT

(kDa)

37

3-pHis

HA

37

IB:

37

3-pHis

H266F

IP: HA

25

20

15

FLAG

37

Total lysate

GST-PHIP-1

IB:

T7-GPC-2

T7-GPC-2

T7-GPC-2T7-GPC-2

HA-GPB-1(WT)

HA-GPB-1(WT)

HA-GPB-1

HA-GPB-1(WT) GST-NDK-1

GST-NDK-1

GST-NDK-1

GST-NDK-1

EE

AA

5A

12 112

122 GST-PHIP-1

122

T H45A

GST-PHIP-1

GST-PHIP-1

- GST-PHIP-1

WT

H45A

WT

W H

(kDa)

(kDa) IB:

(kDa) IB: (kDa)

IB:

3-pHis 37

3-pHis37

3-pHis

37

37

T7-GPC-2

HA-GPB-1(WT)

GST-NDK-1

3-pHis

HA

HA 37

IP: HA

HA

HA

37

37

IP: HAIP: HA IP: HA

37

IP: HA

44

Figure 4.

His-phosphorylation of GPB-1.

His-phosphorylation of GPB-1 in animals. The phip-1(km96) mutant

animals carrying the 3XFLAG::gpb-1 or 3XFLAG::gpb-1(H266F) knock-in

allele were lysed. The lysates were treated with or without heating (95°C)

and immunoblotted (IB) with anti-3-pHis and anti-FLAG antibodies.

NDK-1 phosphorylates GPB-1 in vitro. COS-7 cells were co-transfected

with HA-GPB-1 or HA-GPB-1(H266F) and T7-GPC-2, and cell lysates

were immunoprecipitated (IP) with anti-HA antibodies. Immunopurified

GPB-1 was subjected to the in vitro kinase assay with recombinant GSTNDK-1. Phosphorylated GPB-1 was detected by immunoblotting (IB) with

anti-3-pHis antibodies.

PHIP-1 dephosphorylates GPB-1 in vitro. COS-7 cells were co-transfected

with HA-GPB-1 and T7-GPC-2, and cell lysates were immunoprecipitated

(IP) with anti-HA antibodies. The immunopurified HA-GPB-1 was first

subjected to the in vitro kinase assay with recombinant GST-NDK-1.

Phosphorylated GPB-1 was then equally aliquoted and subjected to the in

vitro phosphatase assay with recombinant GST-PHIP-1 or its variants.

Phosphorylated GPB-1 was detected by immunoblotting (IB) with anti-3pHis antibodies.

45

GPCR

Gβ

His

Gγ

Gα

NDPK

PHPT1

GDP

Gβ

His

Gγ

Gα

Gβ

His

GDP

Gα

GTP

NS

ns

✱✱

ns

NS

Gγ

✱✱

Relative axon length

1.0

0.8

0.6

0.4

0.2

0.0

(n)

WT

goa-1

(km98)

phip-1

(km96)

(54)

(41)

(46)

Goα

goa-1

gpb-1

goa-1

phip-1

(km96); (Q205L) (H266F) (Q205L);

gpb-1

goa-1

(H266F)

(km98)

GOA-1

(47)

(50)

EGL-30

Gqα

EGL-8

PLCβ

(44)

GTP

GTP

PI(4,5)P2

DAG

TPA-1

PKC

JNK pathway

Axon regeneration

(36)

46

Figure 5.

His-phosphorylation of GPB-1 inhibits axon regeneration by

activating GOA-1 Goa.

GPCR-independent Ga activation by His-phosphorylation of Gb. NDPK

phosphorylates Gb, while PHPT1 counteracts this phosphorylation. When

Gb is His-phosphorylated, a high-energy pHis intermediate is transferred

to GDP liganded to Ga, generating a GTP-bound form, which in turn

activates G protein.

Relative axon length 24 h after laser surgery at the young adult stage. The

number (n) of axons examined from three biological replicates is shown.

The black bar in each violin plot indicates the median. *P < 0.05, **P <

0.01, as determined by the Kruskal-Wallis test and Dunn’s multiple

comparison test. NS, not significant.

The relationship between EGL-30 Gqa and GOA-1 Goa in axon

regeneration. EGL-30 activates EGL-8 PLCb, which in turn generates

DAG from phosphatidylinositol bisphosphate [PI(4,5)P2]. DAG activates

TPA-1 PKC, resulting in the activation of the JNK pathway to promote

axon regeneration. GTP-bound GOA-1 antagonizes the EGL-30 signaling

cascade and inhibits axon regeneration. This inhibition is mediated by the

phosphorylation of His-266 in GPB-1 Gb, which leads to the activation of

GOA-1 Goa signaling.

47

DBD

DBD

PHIP-1

PHIP-1

(H45A)

AD

AD

Growth

vector

vector

UNC-51

UNC-51

(H45A) (274-856)

(274-856)

UNC-51

UNC-51

vector

vector

(274-856)

(274-856)

PHIP-1

PHIP-1

ULK2

(Human)

Kinase

1036 aa

A I K S I 41

A I K A I 41

37

37

63% identity

(kinase domain)

UNC-51

Kinase

(C. elegans)

856 aa

UNC-51(274-856)

GFP-UNC-51

WT

KD

FLAG-PHIP-1

(38)

WT

5A 28A T29A S43A T52A S54A S55A S58A S85A S112

T2

(62)

IB:

FLAG

Phos-tag

(kDa)

15

FLAG

GFP

100

No Phos-tag

25 28 29

43

52 54 55 58

10

11

85 112

T S T S T SS S S S

PHIP-1

pHis PPase

116 aa

12

13

48

Figure 6.

UNC-51 phosphorylates PHIP-1 at Ser-112.

PHIP-1 interaction with UNC-51 by yeast two-hybrid assay. The reporter

strain PJ69-4A was co-transformed with expression vectors encoding

GAL4 DBD-PHIP-1(H45A) and GAL4 AD-UNC-51(274-856), as indicated.

Yeast strains carrying the indicated plasmids were cultured on a selective

plate lacking histidine and containing 5 mM 5-aminotriazole for 4 days.

UNC-51 structure. Schematic diagrams of UNC-51 and human ULK2 are

shown. The kinase domain is shown in red. The catalytic lysine and four

flanking amino acids are shown. Identical and similar residues are

highlighted with black and gray shading, respectively. The unc-51(ks49)

mutation is a splice site mutation, which significantly reduces the unc-51

mRNA level.

UNC-51 phosphorylates PHIP-1 at Ser-112. COS-7 cells were cotransfected with Flag-PHIP-1 (WT or mutants) and GFP-UNC-51 [WT or

∆AIKAI (KD)], and cell lysates were analyzed using Phos-tag SDS-PAGE.

Total lysates were immunoblotted (IB) with antibodies, as indicated. Filled

and open arrowheads indicate unmodified and phosphorylated PHIP-1,

respectively. Asterisk indicates non-specific band.

Schematic representation of the ten Ser/Thr residues and domain

structure in PHIP-1.

49

PLM sensory neuron

Regrowth = X

WT

unc-51(ks49)

phip-1(km96)

phip-1(S112A)

unc-51(ks49); phip-1(S112E)

unc-51(ks49); gpb-1(H266F)

NS

ns

✱✱

✱✱

120

Regrowth (µm)

80

40

(n)

WT

phip-1

(km96)

phip-1

(S112A)

phip-1

(S112E)

unc-51

(ks49)

(50)

(54)

(55)

(33)

(39)

unc-51 unc-51

(ks49); (ks49);

gpb-1

phip-1

(S112E) (H266F)

(43)

(42)

50

Figure 7.

UNC-51 promotes axon regeneration by phosphorylating PHIP-

1.

Scheme for axotomy of PLM sensory neurons in C. elegans.

Representative PLM sensory neurons in indicated genotypes 24 h after

laser surgery. Red arrowheads indicate cut sites. Yellow arrows indicate

the tip of axotomized axons. Scale bar, 10 µm.

Length of PLM regrowth 24 h after laser surgery. The number (n) of axons

examined from three biological replicates is indicated. The black bar in

each violin plot indicates the median. *P < 0.05, **P < 0.01, as determined

by the Kruskal-Wallis test and Dunn’s multiple comparison test. NS, not

significant.

51

N2 phip-1(km96)

phip-1(km96)

3XFLAG::gpb-1

66

WT H2

3XFLAG::gpb-1 WT WT

(kDa)

100

(kDa)

100

75

50

IB:

75

IB:

3-pHis

50

37

1-pHis

37

25

20

25

20

15

15

FLAG

37

Total lysate

1 2

Figure EV1.

FLAG

37

Total lysate

His-phosphorylation in animals.

1-pHis levels in animals. The phip-1(km96) mutant animals carrying the

3XFLAG::gpb-1 or 3XFLAG::gpb-1(H266F) knock-in allele were lysed. The

lysates were immunoblotted (IB) with anti-1-pHis and anti-FLAG

antibodies.

The effect of the phip-1(km96) mutation on 3-pHis levels in animals. Wildtype N2 or phip-1(km96) mutant animals carrying the 3XFLAG::gpb-1

knock-in allele were lysed. The animal lysates were immunoblotted (IB)

with anti-3-pHis and anti-FLAG antibodies.

52

GST-PHIP-1

Heat (95℃)

GST-CheA

GST-PHIP-1

WT

- - GST-PHIP-1

Heat

Heat

(95℃)

- + - (95°C)

GST-CheA

- WT

GST-PHIP-1

-- + --

--

p-CheA(kDa)

p-CheA

GST-CheA

12 112

2AS 12E

5AS 1GST-PHIP-1

H4 -S1 - S1

p-CheA

(kDa)

32P

32P

CheA

CheA

32P

Figure EV2.

CBB

32P

CheA

PHIP-1

CBB

11

22

75

32P

75

PHIP-137

(kDa)

75

CheA

75

PHIP-1

5A 112A 112

p-CheA

(kDa)

75

75

GST-CheA

PHIP-1

37

CBB CBB

33

75

75

37

37

CBB

Dephosphorylation of CheA by PHIP-1 in vitro.

GST-CheA was first incubated without GST-PHIP-1 for autophosphorylation.

Autophosphorylated CheA was then equally aliquoted and subjected to the in

vitro phosphatase assay with GST-PHIP-1 or its variants. Phosphorylated CheA

was detected by autoradiography. A heated sample (95°C) was used as a

negative control. Protein input was confirmed by Coomassie Brilliant Blue (CBB)

staining.

53

WT

phip-1(km96)

lgg-2(tm6544)

phip-1(km96); lgg-2(tm6544)

✱✱✱✱

✱✱✱

120

80

Regrowth (µm)

120

Regrowth (µm)

✱✱✱✱

✱✱✱

ns

ns

NS

✱✱

✱✱

UNC-51

80

PHIP-1

Autophagy

40

40

WT

WT

phip-1

phip-1

(km96)

(km96)

phip-1

(km96)

lgg-2

lgg-2

(tm6544)

(tm6544)

(29)

(25)

(25)

WT

phip-1

lgg-2

(km96);

(tm6544)

phip-1

lgg-2

phip-1

(km96); (ktm6544)

(km96);

lgg-2

lgg-2

(ktm6544)

(tm6544)

(n)

(28)

axon regeneration

54

Figure EV3.

UNC-51 regulates axon regeneration via PHIP-1 and

autophagy.

Representative PLM sensory neurons in indicated genotypes 24 h after

laser surgery. Red arrowheads indicate cut sites. Yellow arrows indicate

the tip of axotomized axons. Scale bar, 10 µm.

Length of PLM regrowth 24 h after laser surgery. The number (n) of axons

examined from two biological replicates is indicated. The black bar in each

violin plot indicates the median. *P < 0.05, **P < 0.01, ***P < 0.001, as

determined by the Kruskal-Wallis test and Dunn’s multiple comparison

test. NS, not significant.

Downstream targets of UNC-51. UNC-51 promotes axon regeneration via

phosphorylation of PHIP-1 and autophagy.

55

Gene

gpb-1

gpd-2

gpd-3

gpd-4

unc-51

Gene product

Gβ

Number of colonies

12

GAPDH

ULK homolog

Appendix Table S1.

PHIP-1(H45A) binding proteins identified by yeast

two-hybrid screen.

phip-1(km96)

lhpp-1(km97)

goa-1(km98)

gpb-1(H266F)

crispr RNA

PCR primer for genotyping (forward)

PCR primer for genotyping (reverse)

crispr RNA

PCR primer for genotyping (forward)

PCR primer for genotyping (reverse)

crispr RNA

PCR primer for genotyping (forward)

PCR primer for genotyping (reverse)

crispr RNA

ssDNA

PCR primer for genotyping (forward)

PCR primer for genotyping (reverse)

phip-1(S112A) crispr RNA

ssDNA

PCR primer for genotyping (forward)

PCR primer for genotyping (reverse)

phip-1(S112E) crispr RNA

ssDNA

gcucgcugacaucgccgaugguuuuagagcuaugcu

ttatcacagtgtgagagcattggg

gaagataatgaaacaactgctctactc

aaacucccguuauaucgagcguuuuagagcuaugcu

catctaaacggaccctttctgcc

gcacgcaaatgtttaccttgagg

cauggguuguaccaugucacguuuuagagcuaugcu

gagctgcaccacatacagtgagtg

tacaatagtcgattttcctgattctcc

aauauuaucaugagaauacaguuuuagagcuaugcu

gttcgacattcgtgctgatcaggaacttgcaatgtatt

cttttgataatattatttgcggaatcactagt

tctccagacttccgcacattcatc

ttatcgtgaccagccaatactcctg

aacauucauuucucuaaugaguuuuagagcuaugcu

cattttaaagcagaaatacccagattataatatccactt

cgcgaacgacggatattgaatctccatgtttgagcatagtt

gtggaatccatgtttaattcccagtggaac

gacgctccacaatgtacaatcgtc

aacauucauuucucuaaugaguuuuagagcuaugcu

cattttaaagcagaaatacccagattataatatccactt

cgaaaacgacggatattgaatctccatgtttgagcatagtt

gtggaatccatgtttaattcccagtggaac

gacgctccacaatgtacaatcgtc

aaguucgcucaucuugcugcguuuuagagcuaugcu

PCR primer for genotyping (forward)

PCR primer for genotyping (reverse)

3XFLAG::gpb- crispr RNA

ssDNA

cgtcgacacttccatcagtaccatcctccggagcacgcaccaccacc

agcagcaagatggattacaaagaccatgatggtgactat

aaggatcatgatattgactataaagacgatgacgataa

gagcgaacttgaccaacttcgacaggaggctgaacag

ctgaagtcgcagattcggg

PCR primer for genotyping (forward) aaaacgcgaacaccgaccaggagc

PCR primer for genotyping (reverse) attgctagaccatgctctggaacg

Appendix Table S2.

DNA and RNA sequences.

56

Table 2. Strains used in this study.

Strain

Genotype

KU501

juIs76 II

KU96

phip-1(km96) I; juIs76 II

KU97

juIs76 II; lhpp-1(km97) V

KU98

goa-1(km98) I; juIs76 II

KU1461

phip-1(km96) I; juIs76 II; kmEx1461 [Punc-25::phip-1]

KU1462

phip-1(km96) I; juIs76 II; kmEx1462 [Punc-25::phip-1(H45A)]

KU1463

juIs76 II; kmEx1463 [Punc-25::ndk-1]

KU1464

juIs76 II; kmEx1464 [Punc-25::ndk-1(H118N)]

KU1465

gpb-1(H266F) juIs76 II

KU1466

phip-1(km96) I; gpb-1(H266F) juIs76 II

KU1467

gpb-1(H266F) juIs76 II; kmEx1463 [Punc-25::ndk-1]

KU1468

goa-1(km98) phip-1(km96) I; juIs76 II

KU455

goa-1(Q205L) I; juIs76 II

KU1469

goa-1(Q205L) I; gpb-1(H266F) juIs76 II

KU1343

muIs32 II

KU1470

muIs32 II; unc-51(ks49) V

KU1471

phip-1(km96) I; muIs32 II

KU1472

phip-1(S112A) I; muIs32 II

KU1473

phip-1(S112E) I; muIs32 II

KU1474

phip-1(S112E) I; muIs32 II; unc-51(ks49) V

KU1475

gpb-1(H266F) muIs32 II; unc-51(ks49) V

KU1476

muIs32 II; lgg-2(tm6544) IV

KU1477

phip-1(km96) I; muIs32 II; lgg-2(tm6544) IV

KU1478

3XFLAG::gpb-1 juIs76 II

KU1479

phip-1(km96) I; 3XFLAG::gpb-1 juIs76 II

KU1480

phip-1(km96) I; 3XFLAG::gpb-1(H266F) juIs76 II

KU1481

juIs76 II; unc-51(ks49) V

Appendix Table S3.

Strains used in this study.

57

112S

TCT

112S

TCT

GCG

GAA

phip-1 gene

Wild type

ATG CCG CTC GCT GAC ATC GCC GAT GTG GAT ATC GAC CCG AAA GGA GTT TTC AAG TAC ATC CTG A…

M P L A D I A D V D I D P K G V F K Y I L

km96

ATG CCG CTC GCT GAC ATC GCG ATG TCA GCG ATA TCC ATG TGG ATA TCG ACC CGA AAG GAG TTT TCA AGT ACA TCC TGA…

(14 bp insertion and

M P L A D I A M S A I S M W I S T R K E F S S T S ＊

1-bp substitution)

lhpp-1 gene

Wild type

km97

(4-bp deletion)

ATG TCA AAT GGA AGA GCG GTT AAT GGG TTC CTG CTC GAT ATA ACG …

M S N G R A V N G F L L D I T

ATG TCA AAT GGA AGA GCG GTT AAT GGG TTC CTG - - -

- AT ATA ACG …

M S N G R A V N G F L

266H

CAT

TTT

gpb-1 gene

3XFLAG

goa-1 gene

Wild type

km98

(5-bp deletion)

ATG GGT TGT ACC ATG TCA CAG GAA GAG CGT GCC GCT CTT GAA AGA …

M G C T M S Q E E R A A L E R

ATG GGT TGT ACC A - -

M G C

- - - CAG GAA GAG CGT GCC GCT CTT GAA AGA …

G R A C R S

58

Appendix Figure S1.

Genome editing of phip-1, lhpp-1, gpb-1, and goa-1.

Genomic structure of the phip-1 gene. Exons are indicated by boxes, while

introns and untranslated regions are indicated by bars. The top and

bottom letters indicate nucleotides and corresponding amino acids,

respectively. The phip-1(km96) mutation is a 14 bp insertion (nucleotides

in red) with 1-bp substitution (nucleotide in blue), which causes a

frameshift (amino acids in bold) and premature stop codon (*) in exon 1.

The phip-1(S112A) and phip-1(S112E) alleles are also shown.

Genomic structure of the lhpp-1 gene. The lhpp-1(km97) mutation is a 4bp deletion, which causes a frameshift (amino acids in bold) and

premature stop codon (*) in exon 1.

Genomic st ...