Journal of Cell Science 109, 653-661 (1996)
Printed in Great Britain © The Company of Biologists Limited 1996
JCS7056
653
Human myosin-IXb, an unconventional myosin with a chimerin-like rho/rac
GTPase-activating protein domain in its tail
J. A. Wirth1,2, K. A. Jensen1, P. L. Post1, W. M. Bement1,* and M. S. Mooseker1,3,4,†
1Department
of Biology, and Departments of 2Internal Medicine, 3Cell Biology and 4Pathology, School of Medicine, Yale
University, New Haven, CT 06520, USA
*Present address: Department of Zoology, University of Wisconsin, Madison, WI 53706, USA
†Author for correspondence at address 1 (e-mail: [email protected])
SUMMARY
The full-length primary structure and expression profile of
a novel unconventional myosin heavy chain, human
myosin-IXb, is described. The primary structure of this
myosin predicts a 229 kDa protein that together with its
recently described rat homolog, myr 5, is the ninth class of
myosins to be identified. In comparison to skeletal muscle
myosin-II, the myosin-IXb ‘head’ has two unusual
features: a novel N-terminal domain of 140 amino acids,
which includes a 60 amino acid extension, and a large
insertion of 126 amino acids in the putative actin-binding
site. The ‘neck’ contains four tandemly repeated IQ motifs,
suggesting that this myosin may have four associated light
chains. The ‘tail’ contains a region similar to regions found
in the chimerins, with a putative zinc and diacylglycerol
binding domain, homologous to the regulatory domain of
protein kinase C and a putative GTPase-activating protein
(GAP) domain of the rho/rac family of ras-like G-proteins.
Northern blot analysis of 16 different human tissues
revealed an ~8 kb transcript that is most highly expressed
in peripheral blood leukocytes, with somewhat lower levels
of expression in thymus and spleen, suggesting that myosinIXb is most abundant in cells of myeloid origin. MyosinIXb was also expressed in a number of other tissues at significantly lower levels. Analysis of myosin-IXb protein
expression, using a tail-domain directed antibody, was
performed in HL-60 cells, a human leukocyte cell. MyosinIXb expression increases by 4- to 5-fold upon induced
differentiation of these cells into macrophage-like cells. The
localization of myosin-IXb is also altered upon differentiation. In undifferentiated HL-60 cells, myosin-IXb colocalizes with F-actin in the cell periphery, while in differentiated cells its localization becomes more cytoplasmic,
with the highest levels in the perinuclear region.
INTRODUCTION
with a tail containing domains generally considered to be
specific to proteins involved in signal transduction: a putative
zinc and diacylglycerol binding domain, similar to those found
in protein kinases C (PKC; Olivier and Parker, 1991; Ono et al.,
1988), and a putative GTPase-activating protein (GAP) domain,
most similar to that found in GAP proteins for the rho/rac family
of G-proteins. Myosin-IXb exhibits a limited range of
expression in human tissues, with highest levels in peripheral
blood leukocytes, suggesting a role for this myosin in actinbased processes in myeloid cells.
Recent studies have revealed the existence of a large myosin
superfamily of actin-based motor genes (for review see
Mooseker and Cheney, 1995). Based on sequence comparisons
of the motor or head domains, 9-10 distinct classes in addition
to the well characterized class II (or conventional) and class-I
myosins have been identified thus far (reviewed by Mooseker
and Cheney, 1995). The myosins-IX were initially discovered in
a PCR-based screen for novel myosins (Bement et al., 1994a;
Wirth et al., 1993). Two distinct myosins-IX, termed IXa and
IXb, were identified and partial cDNA sequences of these were
obtained from human cells of colonic origin, human leukocytes,
human liver, and the porcine epithelial cell line, LLC-PK1
(Bement et al., 1994a). In an independent study, a homolog of
human IXb, rat myr 5, has recently been described (Reinhard et
al., 1995). The finding that myosins-IXa and b define a new
myosin class that is apparently widely distributed among the vertebrates prompted us to characterize one of these myosins (IXb)
in greater detail. We report here that like the rat class IX myosin,
myr 5, human myosin-IXb is a fusion of a divergent myosin head
Key words: Myosin-I, Unconventional myosin, GAP, rho, rac,
Leukocyte, HL-60 cell
MATERIALS AND METHODS
Isolation and sequence analysis of human myosin-IXb
cDNAs
Six overlapping cDNA clones were isolated by five rounds of hybridization screening, using as probes the human myosin-IXb PCR product
described by Bement et al. (1994a) and then the 3′-most cDNA
sequences obtained from successive rounds of screening. Initial clones
encoding the 5′ head domain and portions of the tail were isolated from
654
a
1
J. A. Wirth and others
GGGCGGCTCGA
GCAGCGGCGGCGCTGGCAGGCGGTCGTCCGGCCGGGGACCCGGCCCGGGACCGGCGGCGCGCGGCGGCCGAGGCCAGCTCCAGGACACGCGCGCCCCGAGCCTGGGAGGCATGCTGAAGCCAGGCGGCCGGCAGG
ATGAGTGTGAAAGAGGCAGGCAGCTCGGGCCGCCGGGAGCAGGCGGCCTACCACCTGCACATCTACCCCCAGCTGTCCACCACCGAGAGCCAGGCCTCGTGCCGCGTGACTGCCACCAAGGACAGCACCACCTCG
M S V K E A G S S G R R E Q A A Y H L H I Y P Q L S T T E S Q A S C R V T A T K D S T T S
146
281
46
GACGTCATCAAGGACGCCATTGCCAGCCTGCGGCTGGACGGCACCAAATGTTATGTGCTGGTGGAGGTCAAAGAGTCGGGAGGCGAGGAATGGGTGCTGGACGCCAACGACTCGCCTGTGCACCGGGTGCTGCTA
D V I K D A I A S L R L D G T K C Y V L V E V K E S G G E E W V L D A N D S P V H R V L L
416
91
TGGCCCCGGCGGGCACAGGACGAGCACCCTCAGGAGGATGGCTACTACTTCCTGCTGCAGGAGCGCAACGCAGATGGAACCATCAAGTACGTGCATATGCAGCTGGTGGCGCAGGCCACAGCCACCCGGCGCCTA
W P R R A Q D E H P Q E D G Y Y F L L Q E R N A D G T I K Y V H M Q L V A Q A T A T R R L
551
136
GTGGAGCGTGGCCTCCTGCCACGGCAGCAGGCGGACTTTGATGACCTGTGTAACCTTCCTGAGCTAACCGAGGGCAACCTCCTGAAGAACCTCAAGCACCGCTTCCTGCAACAAAAGATCTACACGTACGCGGGG
V E R G L L P R Q Q A D F D D L C N L P E L T E G N L L K N L K H R F L Q Q K I Y T Y A G
686
181
AGCATCCTGGTGGCCATCAACCCCTTTAAGTTCCTGCCCATCTACAACCCCAAGTACGTGAAGATGTATGAGAACCAGCAGCTGGGCAAGCTGGAGCCACACGTCTTCGCGCTGGCCGACGTGGCCTACTACACC
S I L V A I N P F K F L P I Y N P K Y V K M Y E N Q Q L G K L E P H V F A L A D V A Y Y T
821
226
ATGCTCAGGAAGCGCGTGAACCAGTGCATCGTGTATCCGGGTGAGAGCGGCTCCGGCAAGACCCAGAGCACCAACTTCCTCATCCACTGCCTCACCGCCCTCAGCCAGAAGGGCTACGCCAGCGGCGTCGAGAGG
M L R K R V N Q C I V Y P G E S G S G K T Q S T N F L I H C L T A L S Q K G Y A S G V E R
956
271
ACCATCCTGGGTGCCTGTCCTGTGCTGGAAGCTTTTGGAAATGCCAAGACAGCCCACAACAACAACTCCAGCCGGTTTGGGAAATTCATCCAAGTCAGCTACCTAGAGAGTGGCATCGTGAGAGGAGCTGTCGTC
T I L G A C P V L E A F G N A K T A H N N N S S R F G K F I Q V S Y L E S G I V R G A V V
1091
316
GAGAAATATCTGCTTGAAAAGTCTCGCCTGGTGTCTCAGGAGAAGGATGAGAGGAACTACCATGTGTTTTATTATTTGTTACTTGGGGTCAGCGAGGAAGAGCGCCAAGAATTTCAGCTCAAGCAGCCTGAAGAT
E K Y L L E K S R L V S Q E K D E R N Y H V F Y Y L L L G V S E E E R Q E F Q L K Q P E D
1226
361
TATTTCTACCTCAACCAGCATAACTTGAAGATTGAAGATGGGGAGGACCTGAAGCATGACTTTGAGAGGCTCAAGCAGGCCATGGAGATGGTGGGCTTCCTCCCCGCCACCAAGAAGCAGATTTTTGCCGTCCTC
Y F Y L N Q N H L K I E D G E D L K H D F E R L K Q A M E M V G F L P A T K K Q I F A V L
1361
406
TCGGCCATCCTGTACCTGGGCAACGTCACTTATAAGAAGAGAGCTACAGGCCGAGAGGAAGGGTTGGAGGTCGGGCCACCCGAGGTGCTGGACACCCTGTCGCAGCTTCTGAAGGTGAAGCGAGAAATCTTGGTG
S A I L Y L G N V T Y K K R A T G R E E G L E V G P P E V L D T L S Q L L K V K R E I L V
1496
451
GAGGTTCTGACCAAAAGAAAAACGGTGACCGTCAACGACAAGCTTATCCTTCCCTACAGCCTCAGCGAGGCCATCACTGCCCGCGACTCCATGGCCAAGTCTCTGTACAGCGCCCTGTTCGACTGGATTGTGCTG
E V L T K R K T V T V N D K L I L P Y S L S E A I T A R D S M A K S L Y S A L F D W I V L
1631
496
CGGATCAACCACGCACTCCTCAACAAGAAGGACGTGGAAGAGGCAGTCTCGTGCCTGTCCATTGGGGTCCTGGACATCTTCGGGTTTGAAGACTTCGAGAGGAACAGCTTTGAGCAGTTCTGCATCAACTACGCC
R I N H A L L N K K D V E E A V S C L S I G V L D I F G F E D F E R N S F E Q F C I N Y A
1766
541
AATGAGCAGCTGCAGTATTACTTCAACCAGCACATCTTCAAGCTGGAGCAGGAGGAATATCAGGGCGAGGGGATCACGTGGCACAACATCGGCTACACAGACAATGTCGGCTGCATCCATCTCATCAGCAAGAAA
N E Q L Q Y Y F N Q H I F K L E Q E E Y Q G E G I T W H N I G Y T D N V G C I H L I S K K
1901
586
CCCACGGGCCTCTTCTACCTGCTGGACGAGGAGAGCAACTTCCCCCACGCCACGAGCCAGACCCTGCTGGCCAAGTTCAAACAGCAACATGAGGACAATAAGTACTTCCTGGGCACCCCGGTCATGGAGCCAGCT
P T G L F Y L L D E E S N F P H A T S Q T L L A K F K Q Q H E D N K Y F L G T P V M E P A
2036
631
TTCATCATCCAGCACTTCGCAGGGAAGGTGAAATATCAGATCAAGGACTTCCGGGAGAAGAACATGGACTACATGCGGCCAGACATCGTGGCCCTGCTGCGGGGCAGTGACAGCTCCTACGTGCGGGAGCTCATC
F I I Q H F A G K V K Y Q I K D F R E K N M D Y M R P D I V A L L R G S D S S Y V R E L I
2171
676
GGCATGGACCCCGTGGCCGTGTTCCGCTGGGCCGTGCTCCGGGCTGCTATCCGGGCCATGGCAGTGCTTCGGGAGGCCGGACGCCTGCGGGCCGAGAGGGCCGAAAAGGCTGCAGGTATGAGCAGCCCTGGTGCC
G M D P V A V F R W A V L R A A I R A M A V L R E A G R L R A E R A E K A A G M S S P G A
2306
721
CAAAGTCACCCAGAAGAGCTGCCAAGAGGAGCCAGCACCCCTTCGGAAAAACTTTACCGCGATTTGCATAACCAAATGATCAAGAGCATCAAAGGATTGCCCTGGCAGGGCGAGGACCCCCGTAGCCTTCTCCAG
Q S H P E E L P R G A S T P S E K L Y R D L H N Q M I K S I K G L P W Q G E D P R S L L Q
2441
766
TCCCTCAGTCGGCTCCAGAAACCCCGCGCCTTCATCCTGAAAAGTAAAGGTATCAAACAAAAGCAGATCATTCCAAAGAACCTACTGGACTCCAAGTCCCTGAAACTCATCATCAGCATGACTCTGCACGACCGC
S L S R L Q K P R A F I L K S K G I K Q K Q I I P K N L L D S K S L K L I I S M T L H D R
2576
811
ACCACCAAGTCCCTACTGCACCTGCACAAGAAGAAAAAGCCACCAAGCATCAGCGCCCAGTTCCAGACATCCCTTAACAAGCTCTTGGAGGCACTGGGGAAGGCGGAGCCCTTCTTTATCCGCTGCATCCGTTCC
T T K S L L H L H K K K K P P S I S A Q F Q T S L N K L L E A L G K A E P F F I R C I R S
2711
856
AATGCTGAAAAGAAAGAGCTGTGCTTTGACGACGAGCTGGTCCTGCAGCAGCTGCGCTACACCGGCATGCTGGAGACCGTGCGCATCCGGAGGTCAGGGTACAGCGCCAAGTACACGTTCCAGGATTTCACCGAG
N A E K K E L C F D D E L V L Q Q L R Y T G M L E T V R I R R S G Y S A K Y T F Q D F T E
2846
901
CAGTTCCAGGTGCTCCTGCCCAAGGATGCCCAGCCCTGCAGGGAGGTCATCTCCACCCTCCTGGAGAAAATGAAGATAGACAAGAGGAACTACCAGATCGGGAAGACCAAGGTCTTCCTGAAGGAGACGGAGCGG
Q F Q V L L P K D A Q P C R E V I S T L L E K M K I D K R N Y Q I G K T K V F L K E T E R
2981
946
CAAGCCCTGCAGGAGACGCTGCACCGGGAGGTGGTGCGGAAAATCCTGCTGCTGCAGAGCTGGTTCCGGATGGTGCTGGAGCGTCGGCACTTCCTGCAGATGAAGCGGGCCGCCGTCACCATCCAGGCCTGCTGG
Q A L Q E T L H R E V V R K I L L L Q S W F R M V L E R R H F L Q M K R A A V T I Q A C W
3116
991
CGGTCCTACCGGGTCCGGAGGGCGCTGGAGAGGACGCAGGCTGCCGTGTACCTCCAGGCCGCATGGAGGGGCTACTGGCAGCGGAAGCTCTACCGGCACCAGAAACAGAGCATCATCCGCCTGCAGAGCCTGTGT
R S Y R V R R A L E R T Q A A V Y L Q A A W R G Y W Q R K L Y R H Q K Q S I I R L Q S L C
3251
1036
CGGGGGCACCTGCAGCGCAAGAGCTTCAGCCAGATGATCTCGGAGAAGCAGAAGGCAGAAGAGAAGGAGAGGGAAGCCCTGGAAGCCGCAAGAGCAGGCGCTGAGGAGGGCGGACAGGGTCAGGCGGCTGGAGGG
R G H L Q R K S F S Q M I S E K Q K A E E K E R E A L E A A R A G A E E G G Q G Q A A G G
3386
Fig. 1. (a) The nucleotide and translated a.a. sequence of human myosin-IXb (Genbank accession no. U42391). The deduced primary a.a.
sequence is characterized by a number of domains, subdomains and motifs (see text) including an N-terminal novel domain (relative to muscle
myosin-II; highlighted and bold; this includes an N-terminal extension of ~60 a.a.), an insert within the actin-binding site (underlined), a neck
domain (highlighted and underlined; a portion of this sequence was reported previously as an expressed sequence tag, GenBank accession no.
M85411), a chimerin-like domain with PKC and rho/rac GAP homology (white letters on black highlight), and a proline-rich C-terminal region
(broken lines). The ATP-binding site p-loop motif (GESGSGKT, bold) and the primary consensus sequence for the actin binding site (boxed)
are indicated. The region of nucleotide sequence corresponding to the PCR product used for the initial library screen is underlined in bold (see
also Bement et al., 1994a). (b) Box diagram of myosin-IXb demonstrating the relative sizes of the various domains described in a.
a human liver cDNA library (a gift from Dr James M. Anderson, Yale
School of Medicine); clones encoding the 3′ portion of the tail domain
were isolated from a human small intestine cDNA library purchased
from Clontech (Palo Alto, CA). Both strands were sequenced using the
Sequenase 2.0 kit (United States Biochemical, Cleveland, OH)
according to the manufacturer’s instructions. Multiple sequence alignments were performed as previously described (Cheney et al., 1993).
Northern blot analysis of myosin-IXb expression
Northern blots containing equal loadings of poly(A)+ human RNA
from the tissues indicated in Results were purchased from Clontech
(Palo Alto, CA). The blots were probed at high stringency using a
random priming-labeled probe made from a partial length cDNA
clone of 5 kb spanning most of the open reading frame.
Production of myosin-IXb antibodies
Antibodies were raised against two distinct domains of the heavy
chain. The N-terminal portion of human myosin-IXb (amino acids
(a.a.) 3-147), which bears no significant homology to any other known
myosin classes (see Results), and a.a. 1,252-2,022 of the tail domain.
Both domains were bacterially expressed using two different
expression vectors. These were the pGEX vector (Amrad Corp.
Melbourne, Australia) for the production of glutathione S-transferasecontaining fusion protein and the Qia-Express vector (Qiagen Inc.
Chatworth, CA) for production of histidine-tagged fusion protein. Both
were purified by affinity chromatography (on glutathione and Ni
columns, respectively) following the manufacturers’ protocols. For
antibodies to the N-terminal domain, the glutathione S-transferase-
Human myosin-IXB
655
1081
CAGCAGGTAGCTGAGCAGGGGCCGGAGCCAGCGGAGGATGGCGGGCACCTGGCATCGGAGCCTGAGGTGCAGCCAAGTGACAGGTCCCCCCTAGAGCACTCCTCACCTGAGAAGGAGGCCCCAAGCCCAGAGAAG
Q Q V A E Q G P E P A E D G G H L A S E P E V Q P S D R S P L E H S S P E K E A P S P E K
3521
1126
ACTCTCCCACCCCAGAAAACCGTGGCGGCTGAAAGTCACGAGAAAGTCCCCAGCAGCCGGGAGAAGCGTGAGTCGCGTCGGCAAAGAGGGCTGGAGCACGTCAAGTTCCAGAACAAACACATCCAGTCCTGCAAG
T L P P Q K T V A A E S H E K V P S S R E K R E S R R Q R G L E H V K F Q N K H I Q S C K
3656
1171
GAGGAGAGTGCCCTCAGAGAACCTTCCAGAAGGGTCACCCAGGAGCAAGGGGTGAGTCTCCTGGAAGACAAAAAGGAGAGCAGAGAAGATGAAACCCTTCTAGTCGTAGAGACGGAGGCTGAGAACACATCTCAA
E E S A L R E P S R R V T Q E Q G V S L L E D K K E S R E D E T L L V V E T E A E N T S Q
3791
1216
AAGCAGCCCACAGAGCAACCCCAGGCCATGGCAGTTGGCAAGGTCTCTGAAGAAACTGAGAAGACGCTGCCCAGTGGGAGCCCCAGGCCTGGCCAGTTGGAGCGGCCGACCAGCCTGGCCCTGGACAGCAGGGTC
K Q P T E Q P Q A M A V G K V S E E T E K T L P S G S P R P G Q L E R P T S L A L D S R V
3926
1261
AGCCCACCGGCCCCCGGCAGCGCCCCCGAGACCCCCGAGGACAAGAGCAAACCATGTGGCAGCCCAAGGGTTCAGGAAAAGCCCGACAGCCCCGGAGGCTCCACGCAGATCCAGCGGTACCTGGACGCCGAGCGG
S P P A P G S A P E T P E D K S K P C G S P R V Q E K P D S P G G S T Q I Q R Y L D A E R
4061
1306
CTGGCCAGCGCCGTGGAACTGTGGCGGGGCAAGAAGCTGGTGGCCGCCGCCAGCCCTAGTGCCATGCTCAGCCAGTCCCTGGACCTCAGCGACAGACACCGGGCCACAGGGGCCGCCCTCACGCCCACAGAGGAG
L A S A V E L W R G K K L V A A A S P S A M L S Q S L D L S D R H R A T G A A L T P T E E
4196
1351
AGGCGCACCTCCTTCTCCACGAGCGACGTCTCCAAGCTCCTCCCGTCCCTGGCCAAGGCTCAGCCTGCAGCAGAAACCACGGACGGAGAGCGAAGTGCGAAAAAGCCAGCTGTCCAGAAGAAGAAGCCAGGCGAC
R R T S F S T S D V S K L L P S L A K A Q P A A E T T D G E R S A K K P A V Q K K K P G D
4331
1396
GCATCCTCCCTCCCAGACGCAGGGCTGTCCCCGGGCTCTCAGGTCGACTCTAAATCCACGTTTAAGAGGCTTTTTCTGCATAAAACCAAGGATAAAAAATACAGCCTGGAGGGAGCAGAGGAGCTGGAGAATGCA
A S S L P D A G L S P G S Q V D S K S T F K R L F L H K T K D K K Y S L E G A E E L E N A
4466
1441
GTGTCCGGGCACGTGGTGCTGGAAGCCACCACCATGAAGAAGGGCCTGGAAGCCCCCTCCGGACAGCAGCATCGCCACGCTGCAGGTGAGAAGCGCACCAAGGAACCAGGAGGCAAAGGGAAGAAGAACCGAAAT
V S G H V V L E A T T M K K G L E A P S G Q Q H R H A A G E K R T K E P G G K G K K N R N
4601
1486
GTCAAGATTGGGAAGATCACAGTGTCAGAGAAGTGGCGGGAATCGGTGTTCCGCCAGATCACCAACGCCAATGAGCTCAAGTACCTGGACGAGTTCCTGCTCAACAAGATAAATGACCTCCGTTCCCAGAAGACG
V K I G K I T V S E K W R E S V F R Q I T N A N E L K Y L D E F L L N K I N D L R S Q K T
4736
1531
CCCATTGAGAGCTTGTTTATCGAAGCCACCGAGAAGTTCAGGAGCAACATCAAAACGATGTACTCTGTCCCGAACGGGAAGATCCACGTGGGCTACAAGGATCTGATGGAGAACTACCAGATCGTTGTCAGCAAC
P I E S L F I E A T E K F R S N I K T M Y S V P N G K I H V G Y K D L M E N Y Q I V V S N
4871
1576
CTGGCCACTGAGCGTGGCCAGAAGGACACCAACCTGGTCCTCAACCTCTTCCAGTCACTGCTAGATGAGTTCACCCGTGGCTACACCAAGAACGACTTCGAGCCAGTGAAGCAGAGCAAAGCTCAGAAGAAGAAG
L A T E R G Q K D T N L V L N L F Q S L L D E F T R G Y T K N D F E P V K Q S K A Q K K K
5006
1621
CGGAAGCAGGAGCGTGCTGTCCAGGAGCACAACGGGCACGTGTTCGCCAGCTACCAGGTTAGCATCCCGCAGTCGTGCGAGCAGTGCCTCTCCTATATCTGGCTCATGGACAAGGCCCTGCTCTGCAGCGTGTGC
R K Q E R A V Q E H N G H V F A S Y Q V S I P Q S C E Q C L S Y I W L M D K A L L C S V C
5141
1666
AAGATGACCTGCCACAAGAAGTGCGTGCACAAGATTCAGAGCCACTGCTCCTACACCTACGGGAGGAAGGGCGAGCCAGGCGCTGAGCCTGGCCACTTCGGCGTGTGCGTAGACAGCCTGACCAGCGACAAGGCC
K M T C H K K C V H K I Q S H C S Y T Y G R K G E P G A E P G H F G V C V D S L T S D K A
5276
1711
TCGGTGCCCATCGTGCTGGAGAAGCTCCTGGAACACGTGGAGATGCACGGCCTGTACACCGAGGGCCTCTACCGCAAGTCGGGTGCTGCCAACCGCACTCGGGAGCTCCGGCAGGCGCTGCAGACAGACCCCGCA
S V P I V L E K L L E H V E M H G L Y T E G L Y R K S G A A N R T R E L R Q A L Q T D P A
5411
1756
GCAGTCAAGCTGGAGAACTTCCCCATCCACGCCATCACAGGGGTGCTGAAGCAGTGGCTGCGGGAGCTGCCCGAGCCCCTCATGACCTTCGCACAGTACGGCGACTTCCTCCGAGCCGTCGAGCTGCCGGAGAAG
A V K L E N F P I H A I T G V L K Q W L R E L P E P L M T F A Q Y G D F L R A V E L P E K
5546
1801
CAGGAGCAGCTGGCTGCCATCTATGCCGTCCTGGAGCACCTTCCAGAAGCCAACCACAACTCCCTGGAGAGACTCATCTTCCACCTTGTCAAGGTGGCCCTGCTCGAGGATGTCAACCGCATGTCACCTGGGGCG
Q E Q L A A I Y A V L E H L P E A N H N S L E R L I F H L V K V A L L E D V N R M S P G A
5681
1846
GGCCATTATCTTCGCACCCTGCCTCCTGCGCTGCCCTGACAACTCGGACCCGCTGACCAGCATGAAGGACGTCCTCAAGATCACCACGTGCGTGGAGATGCTGATCAAGGAGCAGATGAGGAAATACAAAGTGCT
L A I I F A P C L L R C P D N S D P L T S M K D V L K I T T C V E M L I K E Q M R K Y K V
5816
1891
AAGATGGAGGAGATCAGCCAACTGGAGGCTGCAGAGAGTATCGCCTTCCGCAGGCTTTCGCTCCTGCGACAAAATGCTAACAAGAGCCCCAAGACCCGAGAGCCTGCTGGAGGAGCGGGCCGGCTCTTGACGACC
K M E E I S Q L E A A E S I A F R R L S L L R Q N A N K S P K T R E P A G G A G R L L T T
5951
1936
TCCAGGGTCTCCCCGAGCCCCAGCACGCGAAACCTGGCCCTCGGAAGCTGGCGGAGCGCGGCCCTCAGGACTCGGGGGACAGGGCGGCCAGCCCGGCCGGGCCGCGCAAGAGCCCTCAGGAGGCGGCCGCCGCGC
S R V S P S P S T R N L A L G S W R S A A L R T R G T G R P A R P G R A R A L R R R P P R
6086
1981
CCGGCACGAGAGAGCCCTGCCCAGCCGCCCAGGAGCCGGCCCCGAGTGAGGACAGAAACCCCTTCCCCGTTAAGCTCCGGTCCACCTCCCTCTCGCTCAAATACAGGGATGGCGCCTCTCAGGAGGTGA
P A R E S P A Q P P R S R P R V R T E T P S P L S S G P P P S R S N T G M A P L R R -
6216
TGAAGAGGTACAGTGCCGAGGTCCGGTTAGGAAGGTCGCTGACCGTGCTCCCGAAGGAGGAGAAGTGTCCCCTCGGGACGGCCCCCGCCCTTCGAGGCACCAGGGACACTAGCATAGTACATCGTGTAATGTTAA
GTATGTCGTTATGTTTCCTACACACATGTGTGTGTATGCATATGTACCCATATAGACATACTTAACATTACACGATGTACTATGCTAGCCCGGAATTCGATATCAAGCTTATCGATAC
b
AAA
AAA
novel N-terminal
domain
actin-binding site insert
protein kinase C
regulatory domain
AA
AAAAA
AAA
rac/rho GAP
domain
ATP-binding site
head
proline-rich
neck
containing fusion protein was used as immunogen, and the histidinetagged protein coupled to CNBr-activated Sepharose (Pharmacia, LKB
Biotechnology Inc., Piscataway, NJ) was used for affinity purification.
The same strategy was used for tail-directed antibodies, except that the
histidine-tagged fusion protein was used as immunogen.
Myosin-IXb expression in HL-60 cell
Undifferentiated HL-60 cells, a human myelocytic cell line (a gift from
Dr A. Sartorelli, Yale School of Medicine), were maintained in RPMI
medium (Hazleton Biologics, Lenexa, KS) containing 15% fetal calf
serum (Hyclone, Logan, UT) with 100 i.u./ml penicillin, 100 µg/ml
streptomycin, and 0.25 µg/ml Fungizone (JRH Biosciences, Lenexa,
KS). These cells were not tested for mycoplasma contamination.
Differentiation into macrophage-like cells was induced by addition of
0.1 µM 12-O-tetradecanoylphorbol-13-acetate (TPA) to the medium
for 24 hours. Cell pellets were collected by low speed sedimentation
and washed twice in Tris-buffered saline with 5 µg/ml aprotinin, 1
µg/ml leupeptin, 1 µg/ml pepstatin, and 1 mM phenylmethylsulfonyl
tail
fluoride. A 1% sample of the cells was retained for protein determination (BCA protein assay, Pierce Chem. Co., Rockford, IL). The
remaining cells were processed for SDS-PAGE by rapid suspension in
boiling sample buffer. After boiling for 5 minutes, samples were
sonicated to disrupt DNA. To determine the relative levels of myosinIXb expression before and after differentiation, immunoblot analysis
of SDS-PAGE gels containing equivalent loadings (at several
dilutions) of total HL-60 cell protein was performed using the taildirected antibody and a chemiluminescence western blotting kit
(Boehringer Mannheim, Indianapolis, IL). A horseradish peroxidaseconjugated anti-rabbit secondary antibody (Promega, Madison, WI)
was used for immunodetection. Intensity of the immunobands was
determined by densitometric scanning using a Bio-Rad model 1650
scanning densitometer (Bio-Rad, Richmond, CA).
For immunolocalization, undifferentiated cells were plated onto
acid-washed glass coverslips coated with 7 µg (from an 0.8 mg/ml
stock solution) of Cell Tak (Collaborative Research, Inc. Bedford,
MA) according to the manufacturer’s instructions. The TPA-treated
656
J. A. Wirth and others
cells were plated onto Cell Tak-treated coverslips and allowed to differentiate for 24 hours. The coverslip-grown HL60 cells were
processed for indirect immunofluorescence in six-well plates
following the methods of Hasson and Mooseker (1994). Briefly, coverslips were washed twice in phosphate buffered saline containing 50
mM EGTA (PBSE) at 37°C and fixed for 30 minutes at room temperature with 4% paraformaldehyde in PBSE. Cells were rinsed in
PBSE buffer containing 1% bovine serum albumin (BSA) and 0.1%
Triton X-100, then incubated for 20 minutes with 10% BSA in PBSE.
The coverslips were incubated in primary antibody (either 50 µg/ml
affinity purified anti-myosin-IXb (tail domain) or non-immune rabbit
IgG) for one hour at 37°C, washed with PBSE then incubated with
fluorescein-conjugated donkey anti-rabbit secondary antibody (1:500
dilution; Amersham, Arlington Heights, IL) and 120 nM rhodaminephalloidin (Molecular Probes, Eugene, OR) for 30 minutes at 37°C.
Coverslips were washed with PBSE buffer and mounted onto slides
with Citifluor mounting medium (Citifluor, Canterbury, UK). Images
were acquired on film (Tmax; Eastman Kodak, Rochester, NY) using
a Nikon Diaphot 300 equipped for epifluorescence.
RESULTS
Primary structure of myosin-IXb
The amino acid sequence of myosin-IXb was deduced from the
a
sequence obtained from six overlapping cDNAs isolated from
human liver and small intestine libraries (Fig. 1a). These clones
encompass 6,449 bp of the myosin-IXb transcript and define a
single open reading frame of 6,066 bp encoding a protein of
2,022 a.a. with a Mr of 229×103. Like other myosins, myosinIXb consists of three distinct regions: an N-terminal head or
motor domain, a neck domain containing four tandemly repeated
IQ motifs, and a large C-terminal tail domain of 976 a.a.
Multiple sequence alignments of the myosin-IXb head (a.a.
1-940) with other known myosins demonstrated that human
myosin-IXb is a member of the ninth known distinct class of
myosin (Mooseker and Cheney, 1995). The head contains all
of the sequences considered diagnostic for myosins, including
conserved ATP and actin-binding domains (Fig. 1). However,
it also has at least two unusual features relative to the conventional muscle myosin-II, for which the atomic structure has
been determined (Rayment et al., 1993b). Like the highly
divergent class-III myosins encoded by the ninaC gene of
Drosophila, myosin-IXb has an N-terminal extension of 60 a.a.
Moreover, homology with the head domain of skeletal muscle
myosin-II does not begin until ~a.a. 150 (Fig. 2a). Unlike
ninaC myosins, whose N-terminal extension consists of a
putative kinase domain, the myosin-IXb extension bears no
M9b
ChkM2
MSVKEAGSSGRREQAAYHLHIYPQLSTTESQASCRVTATKDSTTSDVIKD AIASLRLDGTKCYVLVEVKESGGEEWVLDANDSPVHRVLLWPRRAQDEHP 100
M.........................ASPDAEMAAFGEAAPYLRKSEKER IEAQNKPFDAKSSVFVVHPKESFVKGTI..QSKEGGKVTVK.TEGGET.. 70
M9b
ChkM2
QEDGYYFLLQERNADGTIKYVHMQLVAQATATRRLVERGLLPRQQADFDD LCNLPELTEGNLLKNLKHRFLQQK.IYTYAGSILVAINPFKFLPIYNPKY 199
........LTVKEDQ.....VFS................MNPPKYDKIED MAMMTHLHEPAVLYNLKERYA.AWMIYTYSGLFCVTVNPYKWLPVYNPEV 140
M9b
ChkM2
VKMYENQQLGKLEPHVFALADVAYYTMLRKRVNQCIVYPGESGSGKTQST NFLIHCLTALSQKG..........YASGVERTILGACPVLEAFGNAKTAH 289
VLAYRGKKRQEAPPHIFSISDNAYQFMLTDRENQSILITGESGAGKTVNT KRVIQYFATIAASGEKKKEEQSGKMQGTLEDQIISANPLLEAFGNAKTVR 240
M9b
ChkM2
NNNSSRFGKFIQVSYLESGIVRGAVVEKYLLEKSRLVSQEKDERNYHVFYYLLLGVSEEERQE...FQLKQPEDYFYLNQHNLKIEDGEDLKHDFERLKQ 386
NDNSSRFGKFIRIHFGATGKLASADIETYLLEKSRVTFQLPAERSYHIFYQI.MSNKKPEL.IDMLLITTNPYDYHYVSQGEIT.VPSIDDQEELMATDS 337
M9b
ChkM2
AMEMVGFLPATKKQIFAVLSAILYLGNVTYKKRATGREEGLEVGPPEVLD TLSQLLKVKREILVEVLTKRKTVTVNDKLILPYSLSEAITARDSMAKSLY 486
AIDILGFSADEKTAIYKLTGAVMHYGNLKFKQK..QREEQAEPDGTEVAD KAAYLMGLNSAELLKALCYPRVKVGNEFVTKGQTVSQVHNSVGALAKAVY 435
M9b
ChkM2
SALFDWIVLRINHALLNKKDVEEAVSCLSIGVLDIFGFEDFERNSFEQFC INYANEQLQYYFNQHIFKLEQEEYQGEGITWHNIGYT.DNVGCIHLISKK 585
EKMFLWMVIRINQQLD.TKQPRQYF....IGVLDIAGFEIFDFNSFEQLC INFTNEKLQQFFNHHMFVLEQEEYKKEGIEWEFIDFGMDLAACIELIEKP 530
M9b
ChkM2
..PTGLFYLLDEESNFPHATSQTLLAKFKQQHEDNKYFLGTP.....VME PAFIIQHFAGKVKYQIKDFREKNMDYMRPDIVALLRGSDSSYVRELIGMD 678
M...GIFSILEEECMFPKATDTSFKNKLYDQHLGKSNNFQKPKPAKGKAE AHFSLVHYAGTVDYNISGWLEKNKDPLNETVIGLYQKSSVKTLALL.... 623
M9b
ChkM2
PVAVFRWAVLRAAIRAMAVLREAGRLRAERAEKAAGMSSPGAQSHPEELPRGASTPSEKLYRDLHNQMIKSIKGLPWQGEDPRSLLQSLSRLQKPRAFIL 778
...................FATYG...GEAEGGGGKKGG..............KKKGSSF........................................ 647
M9b
ChkM2
KSKGIKQKQIIPKNLLDSKSLKLIISMTLHDRTTKSLLHLHKKKKPPSIS AQFQTSLNKLLEALGKAEPFFIRCIRSNAEKKELCFDDELVLQQLRYTGM 878
..............................................QTVS ALFRENLNKLMANLRSTHPHFVRCIIPNETKTPGAMEHELVLHQLRCNGV 701
M9b
ChkM2
LETVRIRRSGYSAKYTFQDFTEQFQVLLPKDAQ.....PCREVISTLLEK MKIDKRNYQIGKTKVF 939
LEGIRICRKGFPSRVLYADFKQRYRVLNASAIPEGQFMDSKKASEKLLGS IDVDHTQYRFGHTKVF 767
b
954
977
999
1022
REVVRKILLLQSWFRMVLERRHF
LQMKRAAVTIQACWRSYRVRRA.
LERTQAAVYLQAAWRGYWQRKLY
RHQKQSIIRLQSLCRGHLQRKSF
976
998
1021
1044
Fig. 2. (a) The myosin-IXb head domain is highly divergent from that of skeletal muscle myosin-II. Significant findings include a unique Nterminal novel domain and extension (boxed) and a 150 a.a. insert (underlined) that corresponds exactly to the position of a smaller surface
loop (42 a.a.) at the actin-binding site of chicken skeletal muscle myosin-II (see Rayment et al., 1993a,b). (b) The myosin-IXb neck domain
contains 4 IQ motifs; ~23 a.a. tandem repeats shown to be involved in light chain binding (see text for references).
Human myosin-IXB
sequence similarity to other known proteins, except human
myosin-IXa (60% identity, W. Bement, unpublished observations) and a rat homolog of human myosin-IXb, myr5
(Reinhard et al., 1995; see below). Alignments also revealed
the presence of a large (150 a.a.) insertion at precisely the
position of a smaller (42 a.a.) loop that was unresolved in the
crystal structure of the muscle myosin-II head (Fig. 2a;
Rayment et al., 1993b). Assuming that the structure-function
relationships for the head domain of myosin-IXb are similar to
those determined for muscle myosin-II (Rayment et al.,
1993a,b), this large insertion lies within the actin-myosin
contact site, and thus might have an important impact on the
nature of the myosin-IXb-actin interaction. In this regard, we
note that the insertion is highly basic, with a pI of 11.6. With
respect to potential modes of regulation of myosin-IXb
enzymatic activity, we recently noted (Bement et al., 1994a;
Bement and Mooseker, 1995) that almost all known myosins
contain either an acidic residue or a serine or threonine at a
conserved site with the head (a.a. 463 in myosin IXb), which
is shown for amoeboid myosins-I to be the site of heavy chain
phosphorylation (Korn et al., 1988). Myosin-IXb has an
aspartic acid residue at this site, indicating that like most
myosins (and unlike amoeboid myosins-I and probably the
class VI myosins) it is not subject to this mode of regulation.
The neck domain of myosin-IXb (a.a. 940-1045) consists of
four distinctive, ~23 a.a. repeats (Fig. 2b) referred to as IQ
motifs (Cheney and Mooseker, 1992). At least one IQ motif is
found in the neck domain of every myosin sequenced to date,
and they appear to confer light chain binding (reviewed by
Mooseker and Cheney, 1995). Assuming that the ratio of IQ
motifs to light chains is 1:1, myosin-IXb would be predicted
to bind four light chains.
The tail domain of myosin-IXb (a.a. 1,046-2,022), lacks any
regions predicted to form coiled-coil alpha-helical domains
and thus this myosin may be single headed. The N-terminal
most region of the tail consists of ~580 a.a., with no significant sequence similarity to other known proteins except rat myr
5 (see below). Remarkably, however, this region is followed
by a sub-domain (a.a. 1,629-89) similar in primary structure to
657
zinc and phospholipid binding region of PKCs (Ono et al.,
1988; Olivier and Parker, 1991; Fig. 3). Moreover, The PKClike sub-domain is followed by a segment (a.a. 1713-1856)
similar in structure to GAPs for the rho/rac family of ras-like
GTP-binding proteins (for review see Boguski and
McCormick, 1993; Lamarche and Hall, 1994; Hall, 1994). This
GAP domain conforms to the consensus structurally conserved
regions (SCR) SCR1, SCR2, and SCR3 as previously
described (Boguski and McCormick, 1993). The juxtaposition
of the domain similar to the PKC regulatory domain followed
by a putative rho/rac GAP is highly reminiscent of the rac
GAPs, N- and β-chimerins, which also have also have a PKC
regulatory subunit-like domain N-terminal to a GAP domain
(Hall et al., 1990; Diekmann et al., 1991; Leung et al., 1993).
The remaining C-terminal portion (a.a. 1,857-2,022) of the
myosin-IXb tail is proline-rich (18%) but shares no obvious
homology to other known proteins or motifs including the
proline-containing SH3 targeting motifs (Cheadle et al., 1994;
Sparks et al., 1994; Yu et al., 1994) found in some rho/rac
GAPs (reviewed by Lamarche and Hall, 1994).
Primary structure comparison of human myosin-IXb
and rat myr 5
While this study was in the initial review process, the primary
structure of the class IX myosin, rat myr 5 was reported
(Reinhard et al., 1995). A comparison of human myosin-IXb
with myr 5 indicates that these two myosins are quite similar,
although there are several significant differences, raising the possibility that these two myosins may not be orthologs (Fig. 4).
Overall, these myosins exhibit ~82% identity, but the percentage identity varies by subdomain. The head domains are 92%
identical, although the unique N-terminal ~150 a.a. of these
myosins, which includes the N-terminal extension discussed
above are only ~80% identical. Like myosin-IXb, myr 5 has a
neck domain containing 4 IQ motifs (85% identity). A comparison of the tail domains reveals that the respective chimerin-like
GAP domains of both proteins are highly similar to one another
(92% identical). This includes a highly conserved ~150 residue
segment N-terminal (a.a. 1,480-1,629 in myosin-IXb; a.a. 1,440-
PKC Subdomain
155
1629
83
YIKNHEFIATFFGQPTFCSVCKDFVWGLNKQGYKCRQCNAAIHKKCIDKIIGRCTGTAANS 215 PKC-∆
EHNGHVFASYQVSIPQSCEQCLSYIW.LMDKALLCSVCKMTCHKKCVHKIQSHCSYTYGRK........GEPGAEPGHFGVCVDSLTSDKASV...PIV
NFKVHTFRG.....PHWCEYCANFMWGLIAQGVKCADCGLNVHKQCSKMVPNDC................KPDLKHVKKVYSCDLTTLVKAHTTKRPMV
1061 KRERSKVPYI
153 NPEQEPIPIV
rho/racGAP Subdomain
SCR-1
myosin-IXb
N-chim
BCR
rhoGAP
SCR-2
1716
161
1071
163
LEKLLEHVEMHGLYTEGLYRKSGAANRTRELRQALQTDP..AAVKLENF .PIHAITGVLKQWLRELPEPLMTFAQYGDFLRAVELPEKQEQLAAIYAVL
VDMCIREIESRGLNSEGLYRVSGFSDLIEDVKMAFDRDGEKADISVNMY EDINIITGALKLYFRDLPIPLITYDAYPKFIESAKIMDPDEQLETLHEAL
VRQCVEEIERRGMEEVGIYRVSGVATDIQALKAAFDVNNKDVSVMMSEM .DVNAIAGTLKLYFRELPEPLFTDEFYPNFAEGIALSDPVAKESCMLNLL
LRETVAYLQAHALTTEGIFRRSANTQVVREVQQKYNMG...LPVDFDQY NELHLPAVILKTFLRELPEPLLTFDLYPHVVGFLNIDESQRVPATL.QVL
1812
260
1169
259
EHLPEANHNSLERLIFHLVKVALLEDVNRMSPGALAIIFAPCLLRCPDNS DPLTSMKD...VLKITTCVEMLIKEQMRKYKVKMEEISQLEAAESIAFRR
KLLPPAHCETLRYLMAHLKRVTLHEKENLMNAENLGIVFGPTLMRSPE.L DAMAALND...IRYQRLVVELLIKNEDILF....................
LSLPEANLLTFLFLLDHLKRVAEKEAVNKMSLHNLATVFGPTLLRPSEKE SKLPANPSQPITMTDSWSLEVMSQVQVLLYFLQLEAIPAPDSKRQSILFS
QTLPEENYQVLRFLTAFLVQISAHSDQNKMTNTNLAVVFGPNLLWAKDAA ITLKAINP...I...NTFTKFLLDHQGELFPSPDPSGL............
myosin-IXb
N-chim
BCR
rhoGAP
SCR-3
myosin-IXb
N-chim
BCR
rhoGAP
Fig. 3. The human myosin-IXb tail contains a domain with adjacent PKC-like (regulatory subunit) and rho/rac GAP-like homologies as seen in
the chimerin family of proteins (Hall et al., 1990). A multiple sequence alignment demonstrates that human myosin-IXb has high homology
with human PKC-delta (unpublished data, GenBank accession no. gp:z22521; Olivier and Parker, 1991; Ono et al., 1988) and N-chimerin,
rhoGAP and the Philadelphia chromosome breakpoint cluster region gene product (BCR). Cysteine residues within the PKC subdomain,
implicated in zinc-binding, have been highlighted. The structurally conserved regions (SCR 1-3) as previously described for rho/rac GAPs (for
review see Boguski and McCormick, 1993; Lamarche and Hall, 1994) have been boxed.
658
J. A. Wirth and others
1,590 in myr 5) to the chimerin homology domain (a.a. 1,6301,856 in myosin-IXb; a.a. 1,590-1,816 in myr 5) as well as a 60
a.a. segment C-terminal to the GAP domain (ending at a.a.
residues 1,916 and 1,876 in myosin-IXb and myr 5, respectively). However, the portion of the tail between the neck and
the highly conserved chimerin-homology-containing domains of
these myosins differ substantially both in length (myosin-IXb is
~40 a.a. longer due to several insertions positioned along the
length of this segment) and sequence (only 60% a.a. sequence
identity). Although similar in length (~100 a.a.), the C-terminal
tip of the myosin-IXb tail exhibits no significant sequence similarity to that of myr 5.
Tissue expression profile of myosin-IXb
To determine the tissue expression pattern of myosin-IXb,
northern blot analysis was performed at high stringency using
poly(A)+ RNA isolated from 16 different human tissues. This
analysis revealed an ~8 kb transcript that varied widely in
levels of expression among the different tissues examined (Fig.
5 and results not shown). Peripheral blood leukocytes exhibited
the highest levels of expression, while moderate expression
was also detected in thymus and spleen. Spleen contained a
second, minor transcript of ~9.5 kb that was not detected in
other tissues. Given the ratio of granulocytes to mononuclear
cells in peripheral blood (~3:1), these results suggest that
myosin-IXb expression is highest in cells of myelocytic origin;
however, this expression profile does not exclude expression
in lymphocytes as well. Other tissues with minor levels of
myosin-IXb expression include testis, prostate, ovary, brain,
small intestine, and lung. No detectable transcript was
observed in large intestine, placenta, liver, skeletal muscle,
kidney, heart or pancreas (Fig. 5 and results not shown).
Expression of myosin-IXb in HL-60 cells
Given the RNA profile established above, the human myelocytic cell line, HL-60, was selected as a model system with
which to begin examination of myosin-IXb protein expression.
Immunoblot analysis of HL-60 cells and several other human
cell lines (results not shown) using both the head (results not
shown) and tail-directed antibodies detects an immunogen of
appropriate molecular mass, ~230 kDa (Fig. 6). A second,
slightly higher molecular mass immunogen is sometimes, but
M9
MSvkEAGSSG RReqAayHLH IYPQLstteS QaSCRVTATK DSTTSDVIkD aiASLrLDGt KcYVLVEVKE SGGEEWVLDA nDSPVHRVLL WPRRAQdEHP 100
Myr 5 MSahEAGSSG RRrpAtfHLH IYPQLpsagS QtSCRVTATK DSTTSDVIrD vvASLhLDGs KhYVLVEVKE SGGEEWVLDA sDSPVHRVLL WPRRAQkEHP 100
M9
qEDGYYFLLQ ERNADGtIkY vHmQLvAQaT AtrRLVERGL LPRqQADFDD LCNLPELtEg NLLknLKhRF lQQKIYTYAG SILVAINPFK FLPIYNPKYV 200
Myr 5 rEDGYYFLLQ ERNADGsIqY lHvQLlAQpT AacRLVERGL LPRpQADFDD LCNLPELnEa NLLqsLKlRF vQQKIYTYAG SILVAINPFK FLPIYNPKYV 200
M9
KMYENQQLGK LEPHVFALAD VAYYTMLRKr VNQCIVypGE SGSGKTQSTN FLIHCLTALS QKGYASGVER TILGAcPVLE AFGNAKTAHN NNSSRFGKFI 300
Myr 5 KMYENQQLGK LEPHVFALAD VAYYTMLRKh VNQCIVisGE SGSGKTQSTN FLIHCLTALS QKGYASGVER TILGAgPVLE AFGNAKTAHN NNSSRFGKFI 300
M9
QVsYLESGIV RGAVVEKYLL EKSRLVSQEK DERNYHVFYY LLLGVSEEER QEFQLKQPeD YFYLNQHNLk IEDGEDLKHD FERLkQAMEM VGFLPATKKQ 400
Myr 5 QVnYLESGIV RGAVVEKYLL EKSRLVSQEK DERNYHVFYY LLLGVSEEER QEFQLKQPqD YFYLNQHNLn IEDGEDLKHD FERLqQAMEM VGFLPATKKQ 400
M9
IFaVLSAILY LGNVTYKKRA TGReEGLEVG PPEVLDTLSQ LLKVKREiLV EVLTKRKTvT VNDKLILPYS LSEAITARDS MAKSLYSALF DWIVLRINHA 500
Myr 5 IFsVLSAILY LGNVTYKKRA TGRdEGLEVG PPEVLDTLSQ LLKVKREtLV EVLTKRKTiT VNDKLILPYS LSEAITARDS MAKSLYSALF DWIVLRINHA 500
M9
LLNKKDvEEA VSCLSIGVLD IFGFEDFERN SFEQFCINYA NEQLQYYFnQ HIFKLEQEEY QGEGItWHNI gYTDNVGCIH LISKKPTGLF YLLDEESNFP 600
Myr 5 LLNKKDmEEA VSCLSIGVLD IFGFEDFERN SFEQFCINYA NEQLQYYFtQ HIFKLEQEEY QGEGIsWHNI dYTDNVGCIH LISKKPTGLF YLLDEESNFP 600
M9
HATSqTLLAK FKQQHEDNKY FLGTPVmEPA FIIQHFAGkV KYQIKDFREK NMDYMRPDIV ALLRGSDSSY VReLIGMDPV AVFRWAVLRA AIRAMAVLRE 700
Myr 5 HATShTLLAK FKQQHEDNKY FLGTPVlEPA FIIQHFAGrV KYQIKDFREK NMDYMRPDIV ALLRGSDSSY VRqLIGMDPV AVFRWAVLRA AIRAMAVLRE 700
M9
AGRLRAERAE KA.AGmSSPg aqSHpEELPR GAsTPSEKLY RDLHNQmIKS iKGLPWQGED PRsLLQSLSR LQKPRaFiLK SKGIKQKQII PKNLLDSKSL 799
Myr 5 AGRLRAERAE KAeAGvSSPv trSHvEELPR GAnTPSEKLY RDLHNQiIKS lKGLPWQGED PRrLLQSLSR LQKPRtFfLK SKGIKQKQII PKNLLDSKSL 800
M9
kLIISMTLHD RTTKSLLHLH KKKKPPSISA QFQTSLNKLL EALGKAEPFF IRCIRSNAEK KELCFDDELV LQQLRYTGML ETVRIRRSGY SAKYTFQDFT 899
Myr 5 rLIISMTLHD RTTKSLLHLH KKKKPPSISA QFQTSLNKLL EALGKAEPFF IRCIRSNAEK KELCFDDELV LQQLRYTGML ETVRIRRSGY SAKYTFQDFT 900
M9
EQFQVLLPKD aQPCREvIst LLEKmkiDkr NYQIGKTKVF LKETERQALQ EtLHrEVvRk ILLLQSWFRM VLERRHFlQM KrAAvTIQAC WRSYRVRRaL 999
Myr 5 EQFQVLLPKD vQPCREaIaa LLEKlqvDrq NYQIGKTKVF LKETERQALQ ErLHgEVlRr ILLLQSWFRM VLERRHFvQM KhAAlTIQAC WRSYRVRRtL 1000
M9
ERTqAAVYLQ AAWRGYwQRk lYrHQkqSII RLQSLCRGHL QRkSFSQMis EKQKAEeker eAlEaArAga eEGgqgqaAg GqQvaEqgpe PaEDgghLas 1099
Myr 5 ERTrAAVYLQ AAWRGYlQRq aYhHQrhSII RLQSLCRGHL QRrSFSQMmL EKQKAE.... qArEtAgAem sEGepspvAa GeQpsEh... PvEDpesLgv 1093
M9
EpEvqpsdrS PlehsSPeKE aPSPEktlPp QKTVaAESHE KVPSSREKRE SRRQRGLEHV kfQNKHIQSC kEE.SaLREP SRrvtqEqGv SllEDkKEsR 1198
Myr 5 EtEtwmnskS P.nglSPkKE iPSPEmetPa QKTVpAESHE KVPSSREKRE SRRQRGLEHV erQNKHIQSC rEEnStLREP SRkaslEtGe SfpEDtKEpR 1192
M9
EDetllvvET eAentsqkqP teqpqamavg kvseetektl psgsprPgqL eRPtSLaLDS RVSPpaPgSa petPeDksKp cgSprVQeKP dSPgGSTQIQ 1298
Myr 5 EDgletwtET aApscpkqvP .......... ........iv gdpprsPspL qRPaSLdLDS RVSPvlPsSs lesPqDedKg enStkVQdKP eSPsGSTQIQ 1274
M9
RYl..DaERL AsAVElWRGK KLvaaaspSA MLSQSLDLSd rhRatGAALT PTEERRtSFS TSDVSKLlPs lakaqpaaet tDGerSAKKP AvqKKKpgda 1396
Myr 5 RYqhpDtERL AtAVEiWRGK KL.....aSA MLSQSLDLSe kpRtaGAALT PTEERRiSFS TSDVSKLsPv ktste..... vDGdlSAKKP AghKKKsedp 1364
M9
SslPDAGLsp GSQvDSKStF KRLFLHKtKD KKySLEGaEE lEnavSGhvv leattmkKgL eaPSgQQHRH aaGEKrtkep ggKGKKNRNv KiGkITVSEK 1496
Myr 5 SagPDAGLpt GSQgDSKSaF KRLFLHKaKD KKpSLEGvEE tEg..SGgqa aqeaparKtL dvPSsQQHRH ttGEKpl... ..KGKKNRNr KvGqITVSEK 1457
M9
WRESVFRqIT NANELKyLDE FLLNKiNDLR SQKTPIESLF IEATEkFRSN IKTMYSVPNG KIHVGYKDLM ENYQIVVSNL AtERGqKDTN LVLNlFQSLL 1596
Myr 5 WRESVFRkIT NANELKfLDE FLLNKvNDLR SQKTPIESLF IEATErFRSN IKTMYSVPNG KIHVGYKDLM ENYQIVVSNL AaERGeKDTN LVLNvFQSLL 1557
M9
DEFTRgYtKn DFEPVKqsKA QKKKRKQERA VQEHNGHVFA SYQVsIPQSC EQCLSYIWLM DKALLCSVCK MTCHKKCVHK IQShCSYTyg RKgEpGAEPG 1696
Myr 5 DEFTRsYnKt DFEPVK.gKA QKKKRKQERA VQEHNGHVFA SYQVnIPQSC EQCLSYIWLM DKALLCSVCK MTCHKKCVHK IQSyCSYTgr RKsElGAEPG 1656
M9
HFGVCVDSLT SDKASVPIVL EKLLEHVEMH GLYTEGLYRK SGAANRTREL RQALQTDPAa VKLEnFPIHA ITGVLKQWLR ELPEPLMTFA QYGDFLRAVE 1796
Myr 5 HFGVCVDSLT SDKASVPIVL EKLLEHVEMH GLYTEGLYRK SGAANRTREL RQALQTDPAt VKLEdFPIHA ITGVLKQWLR ELPEPLMTFA QYGDFLRAVE 1756
M9
LPEKQEQLAA IYAVLeHLPE ANHnSLERLI FHLVKVALLE DVNRMSPGAL AIIFAPCLLR CPDNSDPLTS MKDVLKITTC VEMLIKEQMR KYKVKMEEIs 1896
Myr 5 LPEKQEQLAA IYAVLdHLPE ANHtSLERLI FHLVKVALLE DVNRMSPGAL AIIFAPCLLR CPDNSDPLTS MKDVLKITTC VEMLIKEQMR KYKVKMEEIn 1856
M9
qLEAAESIAF RRLSLLRQNA nkspKtrepa ggaGrlltts Rvspspstrn LalgswrsAa lrtrgtgrpa rpgraralrr rpprpaResp aqPprSrprv 1996
Myr 5 hLEAAESIAF RRLSLLRQNA pwplKlgfss pyeGvrtksp Rtpvvqdlee LgalpeeaAg gdedrekeil meriqsikee kedityRlpe ldPrgSdeen 1947
M9
rtetpSplss gpppsRsntG mAplrr 2022
Myr 5 ldsetSaste slleeRavrG aAee
1980
Fig. 4. Primary structure comparison of myosin-IXb and rat myr 5. The deduced a.a. sequence of human myosin-IXb and rat myr 5 (Reinhard
et al., 1995) are aligned using the Pileup program of the Wisconsin package (Devereaux et al., 1984). Identical residues are shown in bold.
Human myosin-IXB
not always, observed (e.g. lane 4, Fig. 6). We have also
detected myosin-IXb immunogen in two other human cell lines
tested, HepG2, a liver cell line, and Caco-2BBe, an intestinal
cell line (results not shown). To determine if expression of
myosin-IXb changed during differentiation of HL-60 cells into
macrophage-like cells, quantitative immunoblot analysis was
performed. After TPA-induced differentiation, a 4.7(±2.5 s.d.)fold increase in myosin IXb expression, relative to total cell
protein, was observed (n=5). Although expression is increased,
there is no obvious increase in a protein band at the appropriate molecular mass (Fig. 6, lanes 1 and 2), indicating that
myosin-IXb is not a major protein species in these cells.
Localization of myosin-IXb in both undifferentiated and
macrophage-differentiated HL60 cells was performed by
indirect immunofluorescence (Fig. 7). Undifferentiated cells
are round in shape and exhibit very few cellular projections. In
these cells, myosin-IXb localizes to the cell cortex together
with the bulk of the F-actin, as visualized by phalloidin staining
(Fig. 7a,b). The differentiated cells are larger, and exhibit
Fig. 5. Northern blot analysis of myosin-IXb mRNA transcripts in
human tissues. Lanes loaded with 2 µg of poly(A)+ RNA from
spleen (SP), thymus (TH), prostate (PR), testis (TS), ovary (OV),
small intestine (SI), large intestine (LI) and peripheral blood
leukocytes (PBL) were probed with a 5 kb partial cDNA clone
encompassing the C-terminal half of the head and most of the tail
domain. The migration positions, in kb, of molecular mass markers
are shown on the left.
Fig. 6. Immunoblot analysis of myosin-IXb in undifferentiated and
macrophage-differentiated HL-60 cells. Coomassie Blue staining
(lanes 1 and 2) and immunoblot analysis (lanes 3 and 4) of equal
protein loadings of HL-60 cell homogenates prepared from
undifferentiated cells (lanes 1 and 3) and macrophage-differentiated
cells (lanes 2 and 4). The immunoblot was probed with a taildirected antibody to myosin-IXb. Migration positions of molecular
mass standards are indicated (daltons ×10−3) on the left.
659
numerous cellular extensions. F-actin retains its cortical localization. However, myosin-IXb is more diffusely localized
throughout the cytoplasm, and in many cells, a distinct perinuclear spot of myosin-IXb staining is observed (Fig. 7c-h).
DISCUSSION
The results presented here, together with those of Reinhard et
al. (1995), establish the existence of a novel, ninth class of
myosins that is presumably widely expressed in mammals. The
hallmark feature of human myosin-IXb, which it shares with
rat myr5 is the chimerin-like GAP homology domain in its tail.
Since there is at least one additional class IX myosin gene
expressed in humans and pig (myosin IXa; Bement et al.,
1994b; William Bement, unpublished results) it will be critical
in future studies to determine if such a GAP domain is a
common feature of this myosin class. The structural linkage of
a myosin motor domain to a putative rho/rac GAP is especially
fascinating to consider given the recent studies implicating
both of these proteins in actin dynamics: namely, ruffling and
stress fiber formation (Ridley and Hall, 1992; Ridley et al.,
1992; reviewed by Hall, 1994).
Given the high levels of myosin-IXb expression in leukocytes, it is also important to note that rac has also been implicated as an absolute requirement for generation of superoxide
by NADPH oxidase in human neutrophils. Four proteins,
cytochrome b588, p47 phox, p67 phox, and rac, have been
demonstrated to be the minimum components needed for O2−
production in enzyme assays using recombinant proteins
(reviewed by Bokoch and Knaus, 1994). In the neutrophil
cytosol, rac is complexed with [rho]GDP dissociation inhibitor
(GDI); upon stimulation of the cells rac is translocated to the
plasma membrane and released from [rho]GDI. It will be
important to explore the possible roles for myosin-IXb in either
participation in or regulation of superoxide production.
Although rat myr 5 and human myosin-IXb exhibit high
degrees of sequence similarity within their respective motor
and GAP domains, there are several lines of evidence that
suggest that these proteins are not functional orthologs. As
noted above, these molecules contain regions of significant
sequence divergence, the most striking of which lie at the tips
of their respective tails. Moreover, based on the protein
expression studies cited by Reinhard et al. (1995), myr 5 is
expressed in a number of tissues (testis, lung, brain and liver)
for which we observed either no, or very low levels of, myosinIXb RNA expression. A critical test will be to determine if myr
5 is highly expressed in leukocytes; unfortunately, neither of
our antibodies cross-reacts with rat tissue. One possibility is
that myr 5 and myosin-IXb actually represent RNA splice
variants of the same gene, giving rise to motors differing in
tail-tip domains, which in turn could play a role in differential
subcellular localization. However, human chromosome
mapping studies performed for myr 5 (M. Bähler, personal
communication) and human myosin-IXb (unpublished observations done in collaboration with the laboratory of Nancy
Jenkins, NCI, Fredericksville, MD) suggest that the genes for
these two class IX myosins are found on different chromosomes.
As the first step in dissecting the function of myosin-IXb in
leukocytes, we have analyzed its expression and localization
660
J. A. Wirth and others
Fig. 7. Localization of myosin-IXb in
HL-60 cells. Undifferentiated (a,b)
and macrophage-differentiated HL-60
cells (c-j) were stained with either
rhodamine-phalloidin to visualize Factin (b,d,f,h,j), the tail-directed
myosin-IXb antibody (a,c,e,g), or an
equivalent concentration of
nonimmune rabbit IgG (i). Bar,
10 µm.
in the human myelocytic cell line, HL-60. TPA treatment of
these cells induces their differentiation into macrophage-like
cells. As this occurs, the steady state level of both myosin-IXb
immunogens dramatically increases while myosin-IXb subcellular localization shifts from the F-actin-rich cell cortex to a
more diffuse cytoplasmic distribution; many cells show concentrated staining in a perinuclear spot, which is reminiscent
of Golgi staining. The marked increase in expression is consistent with a role for myosin-IXb in the acquisition and/or
maintenance of the macrophage-like phenotype. The shift from
the actin-rich cortical cytoskeleton to the cytoplasm suggests
that there is a regulated association of myosin-IXb with the
actin cytoskeleton in differentiated HL-60 cells. Of particular
interest is the potential association of myosin-IXb with the
Golgi in differentiating cells. During differentiation, these cells
increase dramatically in size (Fig. 7) and there must be a
resultant increase in cell surface area. This surface area
increase must in turn be accompanied by increased membrane
biogenesis; myosin-IXb might play a G-protein-coupled regulatory and/or transport function in membrane traffic.
Human myosin-IXB
The studies of Reinhard et al. (1995) using bacterially
expressed segments of the myr 5 tail have demonstrated that
the GAP domain and zinc binding domain within the PKC
homology domain are both active. Surprisingly, since
chimerins are rac-specific GAPS (reviewed by Hall, 1994),
these workers demonstrated that the myr 5 tail exhibits its
highest GAP activity for rho (rhoA) not rac (as tested using
rac1). The GAP activity of human myosin-IXb has not been
determined, although it seems likely that its activity will be
similar to that of myr 5 given the high level of sequence
identity between these respective GAP domains. A critical
focus of future study on these myosins will be to assess the
biological activities (actin binding, motor properties and GAP
activity) of the native molecule particularly, to determine what,
if any, interplay between the motor and GAP domains of this
remarkable chimeric molecule might occur.
We are grateful to Martin Bähler for communicating results prior
to publication. We also thank Marc Schwartz for technical assistance
and Richard Cheney for his help with the phylogenetic analysis of the
myosin-IX sequences. J.A.W. was supported by a Winchester Fellowship, Section of Pulmonary and Critical Care Medicine, and a
Boerhinger-Ingelheim Postdoctoral Fellowship. W.M.B. was
supported by a postdoctoral fellowship from the American Cancer
Society. This work was supported by NIH grant DK 25387, NIH
program project grant DK 38979. a Yale Liver Center Pilot Project
grant (DK 34989) and a basic research grant from the MDA.
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(Received 24 October 1995 - Accepted 19 December 1995)