©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Phosphorylation of Kinesin inVivo Correlates with Organelle Association and Neurite Outgrowth (*)

(Received for publication, August 1, 1994; and in revised form, December 5, 1994)

Kyung-Dall Lee Peter J. Hollenbeck (§)

From the Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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, (^1)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.


EXPERIMENTAL PROCEDURES

Cell Culture

All reagents, culture media, and supplements were from Sigma unless specified otherwise. Primary cultures of chick sympathetic neurons and fibroblasts were prepared and grown as described previously(6, 21) . PC12 cells were grown in Dulbecco's modified Eagles medium (DMEM) supplemented with 0.6% glucose, 5% fetal bovine serum, 5% horse serum, 100 units/ml penicillin, and 100 µg/ml streptomycin in a 5% CO(2) incubator. For the induction of neurites, cells were kept continuously in medium containing 50 ng of NGF (2.5 S NGF, Boehringer Mannheim) per ml.

Metabolic Labeling Experiments

Neuronal, fibroblast, and PC12 cultures were labeled with [P]orthophosphate essentially as described previously(21) . Cultures were first washed with phosphate-free DMEM and then incubated for 16-18 h in phosphate-free DMEM containing carrier-free H(3)PO(4) (ICN Biomedicals, Irvine, CA) at 0.5-1 mCi/ml. Differentiated PC12 cells were maintained in NGF for 3-6 days prior to labeling with P. For [S]methionine/cysteine labeling experiments, neuronal cultures were washed 2 times 1 h with methionine-free DMEM and incubated 16-18 h in methionine-free DMEM containing 0.5 mCi/ml [S]methionine and [S]cysteine (protein labeling mix, DuPont NEN) and NGF. After incubation in radiolabel, cells were washed with L-15 medium and lysed with PHEE (65 mM PIPES, 25 mM HEPES, 8 mM EGTA, 1 mM EDTA, pH 7.0) supplemented with 0.6 units/ml aprotinin, protease inhibitor mixture as described previously(31) , 0.1 mM Na(3)VO(4), 10 mM NaF, 10 mM glycerophosphate, 10 mM Na(3)PO(4), 2 mM ATP (PHEE) plus 1% Triton X-100. For the sequential extraction of kinesin from fibroblasts, cells were first extracted with PHEE plus 0.02% saponin with gentle agitation for 4 min at room temperature. Then cells were rinsed with PHEE followed by a second extraction with PHEE plus 1% Triton X-100. For pulse-chase experiments, a 16-18 h incubation with either [P]orthophosphate- or [S]amino acid-containing medium was followed by three washes with L-15, and cells were then restored to normal medium without methylcellulose until the time of lysis.

Isolation of Kinesin by Immunoadsorption

Kinesin was immunoadsorbed from clarified cell lysates or brain supernatants as described previously using SUK4 monoclonal antibody (mAb) against KHC coupled covalently to CNBr-activated Sepharose beads(21, 32) . Cell lysates were clarified by centrifugation for 10 min at 17,000 rpm. To prepare chick brain kinesin, 19-day embryonic chick brains were homogenized in an equal volume of PHEE containing 1% Triton X-100, and the homogenates were clarified by centrifugation at 100,000 times g at 4 °C for 30 min. Supernatants were incubated with SUK4-Sepharose beads with gentle continuous mixing for 2 h at 4 °C. Beads were then washed 5 times with 30 volumes of Tris-buffered saline containing 1 mM ATP, 10 mM NaF, 0.1 mM Na(3)VO(4), 10 mM Na(3)PO(4), and the protease inhibitors listed above; 3 times with the same buffer containing 0.5-0.7 M KCl but lacking protease inhibitors; and 7 times with Tris-buffered saline alone. Kinesin was eluted by resuspending the washed beads in gel sample buffer (125 mM Tris, 2% SDS, 10% glycerol, 10 mM dithiothreitol, pH 6.8), incubating them for 5 min at 95 °C, and pelleting out the Sepharose beads. Using this procedure, KHC co-immunoadsorbs with equimolar KLC, kinectin in substoichiometric amounts, and a phosphoprotein doublet that varies in M(r) between different tissues and cell types (21) .

Phosphatase Treatment of Kinesin

Kinesin immunoadsorbed by SUK4-Sepharose beads was washed as described above and then beads were split into two aliquots. One aliquot was incubated with 100 units of alkaline phosphatase (Boehringer Mannheim) in 100 µl of 50 mM Tris, 100 µM EDTA, pH 8.5, for 30 min at 37 °C. As a control, the other aliquot was incubated either with phosphatase plus inhibitors or without phosphatase entirely, conditions that yielded identical results. The beads were then washed an additional 5 times with Tris-buffered saline.

IEF, SDS-PAGE, Immunoblotting, Fluorography, and Quantification

Two-dimensional gels were run by a procedure similar to that of O'Farrell(33) . The ampholine (Pharmacia Biotech Inc.) mix for IEF was 450 µl of pH 5-8, 150 µl of pH 6-8, and 150 µl of pH 4-6 ampholine in 10 ml of 4% Nonidet P-40 and 55% urea. IEF was run for 16,000 Vbulleth. The second dimensional SDS-PAGE and other SDS-PAGE were at a single concentration (10 or 11% acrylamide, from a 30T:2.8C stock) according to Laemmli(34) . All the two-dimensional gels were run with carbamylyte IEF standard markers (Pharmacia Biotech Inc.), visible as the train of isoelectric forms near the dye front of the gel in Fig. 1A; this ensured the consistency of IEF and expedited the exact alignment and comparison of different two-dimensional gels. Silver staining was performed by the method of Morrissey(35) , developed sufficiently that band densities that were linearly proportional to the amount of protein loaded (r^2 > 0.98) over the range 5-50 ng of bovine serum albumin. Immunoblotting was performed as described previously (6) . KHC was detected using rabbit polyclonal anti-chicken brain KHC (6) at a 1/3000 dilution. mAbs against the KLC (CKLC.3.9C1) and against kinectin (KR160.4), were gifts from M. P. Sheetz (Duke University). We also used an anti-KLC mAb from K. Pfister (University of Virginia). Secondary antibodies were conjugated with either alkaline phosphatase (Bio-Rad) or horseradish peroxidase (Vector Labs, Burlingame, CA). All gels and blots were scanned using a Pharmacia laser densitometer. Radiolabel incorporation into polypeptides was quantified from gels or immunoblots using a Molecular Dynamics PhosphorImager or by excision of bands and corrected Cerenkov counts, as described previously(21) . Specific activities were determined from these measurements combined with mass determinations made using gel densitometry or quantitative immunoblotting with kinesin as a standard(6) . Specific activities of proteins from experimentally-treated cells were compared with controls by Student's t test, with t > t as the criterion for rejection of H(o).


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.




RESULTS

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.



Isoelectric Forms Are Due to Phosphorylation, and Phosphorylation Is Reversible

In order to assess the reversibility of KHC phosphorylation, we subjected immunoadsorbed neuronal kinesin to phosphatase treatment. Since the binding site of SUK4 mAb on the KHC is not near the sites of phosphorylation(21) , phosphatase treatment of antibody-bound kinesin would not be expected to suffer from steric hindrance. Treatment with either alkaline or acid phosphatase resulted in a pronounced redistribution of KHC isoelectric forms from acidic to more basic ones; removal of a phosphate from KHC results in the shift from one isoelectric spot to a less acidic isoelectric spot. The relative intensity of each KHC isoelectric spot before and after alkaline phosphatase treatment is shown in Fig. 2, A and B, respectively. Similar results were seen for the KLC (not shown). This suggested that these isoelectric forms (or charge isoforms) of kinesin were indeed phosphoisoforms generated by different degrees of phosphorylation. This was directly demonstrated by P metabolic labeling using chick sympathetic neuronal cultures (Fig. 2, C and D). All of the isoelectric forms of KHC and KLC were labeled with P as shown by a fluorogram of kinesin separated by two-dimensional PAGE (Fig. 2C). Fig. 2D shows an equal loading of kinesin after alkaline phosphatase treatment. Cerenkov counts of excised gel bands revealed that up to 85% of the P associated with the KHC and KLC was removable in vitro by dephosphorylation with alkaline phosphatase. Alkaline phosphatase treatment rendered both the kinectin and the 70-80-kDa doublet entirely undetectable on fluorograms like the one in Fig. 2D.

Phosphate Turnover in Vivo

Kinesin phosphorylation and dephosphorylation could only play a role in regulating kinesin's behavior in vivo if the turnover of kinesin-bound phosphate were considerably faster than that of the proteins themselves. To evaluate this possibility, we compared kinesin protein turnover to kinesin-bound phosphate turnover in cultured sympathetic neurons using S and P pulse-chase experiments. While the KHC and KLC protein turnover was typical for these cultures (half-lives of 18-20 h versus 36 h for total protein (note also the extreme stability of kinectin, Fig. 3, A and B)), the kinesin-bound phosphates had considerably shorter in vivo half-lives (4-6 h, Fig. 3C). Thus, the total kinesin-bound phosphate turned over 3.5-fold (KLC) or 3.9-fold (KHC) faster than the proteins themselves.


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 (bullet) and kinectin (box), and (B) KHC () and KLC (up triangle) 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 (up triangle) were determined at intervals up to 10 h and are plotted as above (C). These data are the means of two independent experiments. The tvalues shown are calculated from the slopes of the regression lines.



Phosphoisoform Differences between Soluble and Membrane-associated Kinesin

In order to determine whether the phosphorylation state of kinesin correlates with its association with organelle membranes, we examined chicken embryonic skin fibroblast cultures, in which a gentle sequential detergent extraction procedure separates soluble from membrane-associated proteins(6) . Fibroblast cultures were metabolically-labeled with S to increase sensitivity and facilitate quantification and then extracted in 0.02% saponin-containing buffer to remove soluble proteins followed by 1% Triton-containing buffer to release organelle-associated proteins; all cellular kinesin is released by this combination of extractions(6) . Kinesin from the resulting soluble and membrane-associated pools was separately immunoadsorbed by SUK4-Sepharose beads and analyzed by two-dimensional PAGE, fluorography, and quantification of radiolabel (Fig. 4). Fig. 4shows fluorograms from a typical separation of soluble and membrane-associated fibroblast KHC. These cells contain three major and two minor KHC phosphoisoforms, and their distribution in each fraction (Fig. 4, C and D) shows that the membrane-associated kinesin is highly phosphorylated, while the phosphoisoform distribution of soluble kinesin is skewed toward less phosphorylated states. In particular, KHC phosphoisoform 5 (the most acidic, and thus likely the most highly phosphorylated) is the predominant one in organelle-associated kinesin, amounting to 60% of the total organelle-associated KHC. In the soluble kinesin pool, the fraction of this phosphoisoform is reduced more than 2-fold, while phosphoisoform 3 shows a corresponding increase.


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.).



Regulation of Kinesin Phosphorylation in PC12 Cells by NGF

Next, we investigated whether cells with different amounts of anterograde organelle transport have different levels of kinesin phosphorylation. PC12 cells with and without NGF treatment satisfy this requirement; in presence of NGF, PC12 cells send out and continuously extend neurites, which become several times as long as the cell body diameter after 3 days of treatment and show abundant organelle transport(38) . It has been demonstrated that kinesin is necessary for neurite extension in several systems, including cultured PC12 cells and adult nerve by antisense oligonucleotide treatment and antibody blocking experiments(39, 40, 41) . (^2)If kinesin is recruited to the organelle surface by phosphorylation, then NGF-differentiated PC12 cells, with their large increase in anterograde transport, would show increased kinesin phosphorylation. We investigated this directly by metabolically labeling cells with P either with or without prior NGF differentiation and then quantifying the P specific activity of total cellular KHC, KLC, and co-immunoadsorbing phosphoproteins. Fig. 5shows a typical P labeling pattern of immunoadsorbed kinesin and kinesin-associated phosphoproteins (KAPPs, see (37) ) separated by one-dimensional SDS-PAGE and fluorographed. Both KHC and KLC are labeled with P, along with two co-immunoadsorbing KAPPs of approximate molecular mass of 100 and 80 kDa. As shown in the histogram on the right in Fig. 5, NGF differentiation produced a significant increase in the P specific activity of both the KHC and KLC. However, the KAPPs from PC12 cells did not show a significant change in specific activity when treated with NGF, indicating that under our conditions, NGF treatment does not produce a global increase in protein phosphorylation. The phosphorylation of KHC in NGF-treated PC12 cells was analyzed further by two-dimensional PAGE in order to assess whether the increase of P specific activity described above corresponded to a difference in the phosphoisoform distribution. The KHC phosphoisoform distributions differed between these two conditions in one significant way: the most acidic phosphoisoform increased in proportion with NGF differentiation by more than 2-fold, from 4.1% of the total KHC without NGF to 9.1% with NGF treatment (average of two independent experiments).


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.




DISCUSSION

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.

Kinesin Phosphoisoforms in Vivo

The data presented in Fig. 1and Fig. 2make it clear that kinesin is phosphorylated in vivo at several sites. This is consistent with the complex phosphopeptide patterns seen when kinesin from P-labeled neurons was subjected to partial proteolytic digestion(21) . While KHC and KLC from whole brain consistently show major phosphoisoforms with five different pI values, we also observed cytotypic variation in their composition; sympathetic neurons showed four major phosphoisoforms, while epidermal fibroblasts showed three. Although it is possible that single cell types, and certainly whole tissues, contain more than one kinesin isoform derived from differential mRNA splicing (42) and/or express more than one member of the kinesin gene superfamily(1, 2, 43) , SUK4 mAb, used to isolate kinesin throughout this study, appears to immunoadsorb only one species of conventional kinesin comprising several phosphoisoforms. This is indicated by the lack of molecular weight heterogeneity of both the KHC and KLC and by the results of phosphatase treatment on the isoelectric form distribution of both chains; P labeling is removed from all of the kinesin isoelectric forms, reducing their number virtually to one isoelectric form for each chain.

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),^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.

Phosphorylation and the State of Kinesin in the Cell

Because the phosphorylation sites reside at some distance from the mechanochemical head region of the KHC(21) , we think it is unlikely that phosphorylation directly affects kinesin motor activity, but the recruitment between soluble and organelle-associated states presents another possibility for regulation(6, 21, 30) . The striking difference between the phosphoisoform distributions of soluble and organelle-associated kinesin from fibroblasts is consistent with this possibility; organelle-associated KHC is phosphorylated to the highest levels with its phosphoisoform distribution dominated by the most acidic form, while soluble KHC has a distinct distribution indicating considerably reduced phosphorylation (Fig. 4). The conditions used to solubilize proteins from cells and to immunoadsorb kinesin were designed to maintain the relative distribution of the kinesin phosphoisoforms in vivo as unperturbed as possible(6, 21) . However, considering the imperfect separation of soluble proteins from membrane-associated proteins using the sequential extraction procedure, the difference in the phosphoisoform distribution between soluble and membrane-bound kinesin in cells is probably underestimated in the histograms in Fig. 4. It is probable that as much as 10% of the soluble kinesin is left after saponin extraction and carried into the Triton fraction, while a similar amount of organelle-associated kinesin may contaminate the saponin fraction(6) . Thus, it is possible that the most phosphorylated form of the KHC, phosphoisoform 5 (comprising nearly two-thirds of the Triton fraction) is virtually the only phosphoisoform that is organelle-bound in vivo. Likewise, the error inherent in the procedure would allow that the in vivo soluble pool could be composed entirely of lower phosphorylation states of the KHC.

Kinesin Phosphorylation and the Induction of Anterograde Transport

Finally, we analyzed kinesin phosphorylation before and after induction of neurite elongation in PC12 cells, a condition that produces an increase in anterograde organelle traffic to support developing processes(38, 44, 45) . Here, despite being limited to analysis of total cellular kinesin, we saw that NGF induced not only neurite outgrowth but also a 1.5-2-fold increase in the P specific activity of kinesin along with a 2-fold increase in the relative abundance of the most phosphorylated KHC phosphoisoform. This was not attributable to a global increase in phosphoprotein specific activity since the two KAPPs characteristically seen in PC12 cells underwent no significant change (Fig. 5). By analogy with fibroblast kinesin, the increased phosphorylation of total KHC in PC12 cells is likely to reflect a shift away from the soluble state and toward organelle association.

The Pattern and Role of Kinesin Phosphorylation

In neurons metabolically labeled with P, we could not detect an isoelectric form that did not incorporate label, indicating that at steady state in vivo, all kinesin isoelectric forms are phosphorylated. Our data indicate further that additional phosphorylation, in particular the production of the most acidic phosphoisoform, is coupled to membrane association. Thus, it seems likely that a kinase phosphorylates KHC at some serine residues constitutively upon synthesis and that the putative signals that recruit kinesin to membranes induce additional phosphorylation. Furthermore, these in vivo data suggest caution in the interpretation of in vitro binding experiments employing kinesin and isolated organelle membranes(20, 24, 25) . The phosphorylation state of kinesin used for in vitro binding experiments could be critical; if the majority of kinesin is in a less phosphorylated soluble state, the parameters of kinesin binding obtained using a typical kinesin preparation may not accurately represent the in vivo situation. In addition, although Sato-Yoshitake et al.(20) have demonstrated a potential role for KHC phosphorylation in membrane association by showing that cAMP-dependent phosphorylation of kinesin in vitro affects its membrane binding, our result is the opposite of what they observed in vitro; their data indicated a weaker association of phosphorylated kinesin with vesicles. This may be the result of the use of protein kinase A to phosphorylate kinesin in vitro, since this kinase is apparently not involved in phosphorylating neuronal kinesin in vivo(21) . This issue has also been raised recently by Kotz and McNiven (19) with respect to the body of work on the behavior of pigment granules in chromatophores. In recent studies, McIlvain et al.(37) have suggested a different avenue of regulation; they found that in vitro hyperphosphorylation of KAPPs, but not kinesin itself, correlated with increased motor activity of the kinesin complex in a microtubule-gliding assay. Here, we observed that NGF treatment of PC12 cells up-regulated kinesin phosphorylation without any accompanying hyperphosphorylation of KAPPs (Fig. 5). Whether motility-related hyperphosphorylation of KAPPs occurs in vivo remains to be seen.

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.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant NS27073 and the Harvard Mahoney Neuroscience Institute (KDL). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Neurobiology, Harvard Medical School, 220 Longwood Ave., Boston, MA 02115. Tel.: 617-432-0881; Fax: 617-734-7557; phollenb{at}warren.med.harvard.edu.

(^1)
The abbreviations used are: KHC, kinesin heavy chain; KLC, kinesin light chain; NGF, nerve growth factor; DMEM, Dulbecco's modified Eagles medium; PIPES, 1,4-piperazinediethanesulfonic acid; KAPPs, kinesin-associated phosphoproteins; IEF, isoelectric focussing; PAGE, polyacrylamide gel electrophoresis.

(^2)
K.-D. Lee and P. J. Hollenbeck, unpublished results.


ACKNOWLEDGEMENTS

We would like to thank Rosie Weld for preparation of sympathetic neuronal cultures, Myrta Otero for technical assistance, and Dr. Marguerite Olink-Coux, Kelly Overly, and Robert Morris for helpful discussions. We also thank Mike Sheetz and Kevin Pfister for the gift of antibodies.


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