(Received for publication, August 1, 1994; and in revised form, December 5, 1994)
From the
The motor protein kinesin is implicated in organelle movement
toward the plus ends of microtubules, but little is known about its
interaction with organelle membranes or about the physiological role of
the phosphorylation of kinesin and its associated protein kinectin seen
in neurons in vivo (Hollenbeck, P. J. (1993) J. Neurochem. 60, 2265-2275). Here we have demonstrated that the kinesin
heavy chain (KHC), light chain, and kinectin isolated from chick brain
or sympathetic neurons exist in several isoelectric forms. Metabolic
labeling followed by phosphatase treatment showed that these are
phosphoisoforms, and that phosphorylation is reversible in
vitro. To assess the capability of phosphorylation to regulate
kinesin's state and/or activity, we performed P and
S pulse-chase experiments with neuronal cultures and
determined that kinesin-associated phosphate turns over 3-4 times
faster than the proteins themselves. When the phosphoisoform
distributions for different kinesin pools were analyzed, it was found
that membrane-associated KHC contained predominantly the most highly
phosphorylated isoform, while soluble kinesin consisted of less
phosphorylated KHC isoforms. Nerve growth factor-induced neurite
outgrowth in PC12 cells was found to increase significantly
kinesin's
P specific activity while doubling the
relative abundance of the most highly phosphorylated KHC isoform. These
results demonstrate that the phosphorylation state of kinesin is
closely coupled to its organelle binding and to the magnitude of
organelle transport in the cell. We propose that the phosphorylation
state of kinesin and associated proteins may regulate motility via
association with organelle membranes and, specifically, that KHC
phosphorylation induces membrane association.
A substantial body of evidence from in vitro and in vivo studies has indicated that the motor protein kinesin is involved in organelle transport and membrane traffic (for review, see (1, 2, 3, 4) ). However, little is known about how kinesin's activity is regulated, particularly about the modulation of its association with organelle membranes. Toyoshima and co-workers (5) have identified a 160-kDa integral membrane protein, ``kinectin,'' and proposed that it binds to the tail region of the kinesin molecule, thus serving as a receptor molecule on the organelle surface. However, it is not yet clear whether kinectin is the sole participant in kinesin binding to the organelle membrane or whether additional factors are involved. Moreover, the most important element, the molecular mechanism serving to regulate the partitioning, or interconversion, between soluble and membrane-bound states of kinesin in vivo, remains unknown.
Kinesin is generally recovered as a soluble protein from cell homogenates, and it has been shown by differential detergent solubilization of plasma and organelle membranes that approximately two-thirds of the total kinesin in fibroblasts is in a soluble state while the remaining third is organelle-bound(6) . However, a simple interconversion governed by a single binding affinity constant between kinesin and organelle membranes is unlikely to satisfy the complex requirements of spatiotemporal modulation of organelle transport in cells. Thus, we expect the affinity between kinesin and membranes to be regulated by a mechanism that can respond to the immediate physiological conditions of the cell and alter the state and/or activity of kinesin.
One potential mechanism for this regulation is protein phosphorylation, which is known to play a role in the regulation of nonmuscle myosins (7) and may also be involved in the case of axonemal dynein(8, 9, 10) . In addition, it has been implicated in the regulation of organelle motility in neurons (11) and clearly associated with regulation of pigment granule motility in teleost pigment cells of several kinds (12, 13, 14, 15, 16, 17, 18, 19) . Recent studies indicate that kinesin is a phosphoprotein (20, 21, 22) and that phosphorylation may play a role in regulating its association with organelle membranes or its ATPase activity in vitro. However, direct correlation between the state or motility of kinesin and its phosphorylation has not yet been shown in vivo; it is notable, for instance, that despite the detailed physiological characterization of pigment granule movement and the apparent role of kinesin there(23) , the relationship between regulatory phosphorylation and the motor protein(s) present remains unknown.
Several recent studies have addressed the interaction of purified kinesin with isolated membrane vesicles(20, 24, 25) . Varied membrane preparations have been employed, and all are reported to bind kinesin with a high affinity. However, if the phosphorylation state of kinesin plays a regulatory role in membrane association, the in vivo situation may be considerably more complex than that represented by in vitro binding experiments that employ kinesin preparations with undefined phosphorylation states. Moreover, although it has been shown that in vitro phosphorylation by protein kinase A catalytic subunits alters the affinity of kinesin for membranes(20) , the relevance of these experiments depends upon the identity of the kinesin kinase(s), which is as yet unknown and seems unlikely to be protein kinase A in vivo(21) . Thus, in this report we have further investigated the phosphorylation of kinesin in vivo and addressed whether that phosphorylation is correlated with its association with membranes.
We have
previously established from metabolic labeling studies in diverse cell
types that all three components presumed to be involved in the
kinesin-organelle interaction, KHC, ()KLC, and kinectin, are
phosphoproteins in vivo and that the KHC is phosphorylated
exclusively on serine residues in a <5-kDa region outside of the
N-terminal mechanochemical and microtubule-binding head
domain(21) . Since the C-terminal tail domain of KHC, perhaps
in association with KLC, is thought to be involved in the interaction
with membrane and/or with
kinectin(5, 25, 26, 27) , the
phosphorylation sites are likely to be in the general region of
kinesin-membrane interaction. This is consistent with the possibility
that kinesin-based motility is regulated by a phosphorylation-mediated
interconversion of kinesin between soluble and membrane-bound states.
This idea has been developed previously for myosin, cytoplasmic dynein,
and kinesin by several
authors(6, 21, 28, 29, 30) .
Here, in order to address this hypothesis, we have employed two-dimensional gel electrophoresis and metabolic labeling of cultured cells in order to detect different phosphoisoforms of kinesin and investigate the phosphorylation state of kinesin in its soluble versus membrane-associated forms. Our results show that the KHC and KLC exist, with cytotypic variation, in at least three different isoelectric forms (i.e. phosphoisoforms) due to different phosphorylation states and that the phosphorylation is reversible both in vitro and in vivo. We further demonstrate a striking difference in the phosphorylation states of the KHC between the soluble and membrane-bound kinesin pools. Finally, we show in vivo that kinesin becomes more phosphorylated when anterograde organelle transport is up-regulated by nerve growth factor (NGF)-induced neurite extension in PC12 cells. Based upon these data, we propose that a fraction of cellular kinesin is in an inactive, soluble state, and can be recruited by protein phosphorylation into a form competent to bind organelles with higher affinity and support transport.
Figure 1:
KHC, KLC, and kinectin exist in several
isoelectric forms. A, a silver-stained two-dimensional gel of
kinesin immunoadsorbed from 19-day embryonic chick brain shows several
isoelectric forms of the KHC, KLC, and kinectin. The positions of
molecular mass standards are indicated at the left, the pH
gradient of the isoelectric focusing gel is indicated from left to right along the top, and the carbamylyte
standards used to put different two-dimensional gels in register are
visible as a chain of spots across the lower portion
of the gel (arrow). The largespots at
50 kDa (pI values between 7 and 6.7) are fragments of reduced
immunoglobulins from SUK4 beads. Immunoblots prepared by transfer of a
gel loaded and run identically to the one in A and probed with
rabbit anti-KHC antibody (B), mouse anti-KLC mAb CKLC.3.9C1
confirm the identity of the isoelectric forms as KHC and KLC,
respectively.
Kinesin heavy chain, which was immunoadsorbed from Triton X-100-containing homogenates using SUK4 anti-KHC mAb(36) , co-adsorbed with equimolar KLC along with substoichiometric amounts of kinectin and an unidentified phosphoprotein doublet of 70-80 kDa(5, 21, 37) . Two-dimensional gel electrophoresis of the immunoadsorbed proteins showed that kinesin from embryonic chicken brain was composed of isoelectric forms with differing isoelectric points (Fig. 1A). Both the KHC and KLC were comprised of five major isoelectric forms, while kinectin showed three. The identities of each of these isoelectric forms as bona fide KHC (Fig. 1B), KLC (Fig. 1C), and kinectin (not shown) were confirmed by immunoblotting of two-dimensional gels identical to Fig. 1A. Kinesin isolated from pure cultures of chick sympathetic neurons contained four major KHC isoelectric forms (Fig. 2).
Figure 2:
Isoelectric forms of the KHC are the
result of different degrees of phosphorylation. Kinesin from chick
sympathetic neurons was immunoadsorbed, divided into two equal
aliquots, treated with or without active alkaline phosphatase, and then
subjected to two-dimensional SDS-PAGE. The distribution of KHC
isoelectric forms without (A) and with (B)
phosphatase treatment is shown quantitatively in histograms. Identical
treatment of kinesin immunoadsorbed from P metabolically
labeled sympathetic neurons gave the fluorograms shown in C (untreated) and D (phosphatase treated). Arrowheads denote the positions of corresponding phosphoisoforms of both the
KHC and KLC between phosphatase-treated and untreated samples; these
were determined by precise alignment of two-dimensional gels using
carbamylyte standards as described under ``Experimental
Procedures.'' The phosphoproteins above and to the right of the KHC and above and to the left of the KLC are kinectin, and the 70-80-kDa doublet,
respectively. These are typical data from one of three independent
experiments performed.
Figure 3:
The phosphate associated with the KHC and
KLC turns over several times faster than the proteins themselves do in
neurons. Sympathetic neurons were metabolically labeled with
[S]methionine and
[
S]cysteine overnight and then chased with
medium containing cold amino acids. The
S specific
activities of (A) total protein (
) and kinectin
(
), and (B) KHC (
) and KLC (
) were
determined at intervals up to 48 h and are expressed as percentages of
the specific activities at the zero chase time point. In parallel
experiments, neurons were labeled with
P overnight and
chased with medium containing cold phosphate. The
P
specific activities of KHC (
) and KLC (
) were determined
at intervals up to 10 h and are plotted as above (C). These
data are the means of two independent experiments. The t
values shown are calculated from the slopes of the
regression lines.
Figure 4:
The distribution of KHC phosphoisoforms
differs between soluble and membrane-bound kinesin pools. Fibroblasts
were sequentially extracted to separate soluble from
organelle-associated kinesin as described under ``Experimental
Procedures.'' Kinesin from each fraction was immunoadsorbed and
analyzed by two-dimensional PAGE. The amount of KHC in each
phosphoisoform was then determined by quantitative immunoblotting or by
use of S metabolically labeled cells combined with
fluorography. (A) and (B) show typical fluorograms of
KHC separated by two-dimensional PAGE. A is the kinesin
immunoadsorbed from the soluble fraction, and B is the kinesin
immunoadsorbed from the organelle-associated fraction. Arrowheads denote the three most prominent phosphoisoforms (3, 4, 5) detected in fibroblasts. The
percent distributions of phosphoisoforms in the soluble (C)
and organelle-associated pools (D) are shown in histograms (n = 3, errorbars = 1
S.E.).
Figure 5:
Differentiation of PC12 cells into
neurite-bearing cells by NGF treatment is accompanied by an increase in
the P specific activity of the KHC and KLC, but not of
KAPPs. A representative fluorogram of kinesin immunoadsorbed from
lysates of
P metabolically labeled PC12 cells is shown on
the left. Kinesin was immunoadsorbed from cells labeled for
16-18 h with
P as described under
``Experimental Procedures,'' separated by SDS-PAGE, and
analyzed by quantitative immunoblotting using polyclonal anti-KHC. The
P specific activity of the KHC from NGF-treated (hatchedbars) and untreated (openbars) cells was determined and compared, as shown in the
histogram at right, in which a value of 100 indicates the
specific activities in untreated cultures (n = 3, errorbars = 1 S.E.). Control cells were
handled the same way, except that NGF was omitted, and significant
differences from control values at p < .05 are indicated by asterisks.
In this study, we sought to determine whether the characteristics of kinesin phosphorylation in vivo were reconcilable with a role in the regulation of organelle transport. We analyzed several properties of kinesin phosphorylation in live cells (its complexity, reversibility, turnover, differential distribution between soluble and organelle-associated pools, and response to up-regulation of anterograde transport) and found all to be consonant with a role in regulation; in particular, with a role in the recruitment of kinesin between the soluble and organelle-associated states.
In order
for phosphorylation and dephosphorylation to regulate the function of
kinesin, at least some of the phosphate must be labile relative to the
kinesin polypeptides in vivo. Thus, to compliment our in
vitro evidence for the reversibility of kinesin phosphorylation,
we performed the pulse-chase experiments illustrated in Fig. 3.
These demonstrate that the turnover of total kinesin-bound phosphate is
3.5-4-fold faster than the turnover of the proteins themselves.
Thus, kinesin is not just constitutively phosphorylated at static sites
upon synthesis but shows dynamic phosphorylation behavior. Since all
isoelectric forms of kinesin are phosphorylated, and phosphatase
treatment suggests that some are less labile than others (Fig. 2), this global average is probably an
underestimate of the in vivo turnover rates for the most
labile kinesin phosphorylation site(s). In toto then, these
data indicate that phosphate turnover is sufficiently rapid to fit a
variety of models for the regulation of kinesin activity.
The phosphorylation of KHC may directly induce stronger affinity for the receptor sites on organelle membranes or it may cause a conformational change in kinesin that exposes the membrane binding region of the molecule. The regulation of kinesin ATPase by two different conformational states has been proposed previously by Hackney et al.(30) , who showed that kinesin undergoes a conformational change from a folded to an extended state as ionic strength is increased. If this transition is regulated by intramolecular electrostatic interactions, it seems probable that phosphorylation has a similar effect to that of increased ionic strength, favoring an extended form that can bind to membranes and possesses a higher motor activity. The exact molecular event that leads to the increased phosphorylation of KHC and membrane-association is not yet clear, and it remains possible that kinesin phosphorylation and membrane binding may be indirectly coupled by yet another mechanism(s). Further experiments such as identification of the kinesin-specific kinase and inhibition of kinesin phosphorylation in vivo are needed to elucidate the detailed mechanism.
The role of KLC phosphorylation in the interaction of kinesin with membranes was not directly examined in this report for lack of appropriate reagents to detect KLC in soluble and membrane-associated pools of the fibroblast system. A recent in vitro study indicates that KLC phosphorylation, in conjuction with calmodulin binding, regulates the functions of the KHC head domain(22) , although possible effects on organelle-binding were not examined. Particularly relevant to the potential involvement of KLC phosphorylation in the interaction between kinesin and membranes is the recent finding that the KHC homodimer binds to organelle membranes in vitro in a fashion similar to the conventional native kinesin heterotetramer of KHC and KLC(25) . In light of these studies, our in vivo data showing increased phosphorylation of KLC along with KHC in NGF-treated PC12 cells could reflect the regulation of kinesin motor activity rather than, or in conjunction with, organelle binding.