0 1994 by The American Society for Biochemistry and Molecular Biology, Inc. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 269, No. 29, Issue of July 22, pp. 1917619182, 1994

Printed in U.S.A.

Regulation of Kinesin Activity by Phosphorylation of Kinesin-associated Proteins*

(Received for publication, March 25, 1994)

James M. McIlvain, Jr.S, Janis K. Burkhardtgn, Sarah Hamm-AlvarezSII, Yair Argon$§, and Michael P. SheetzS From the Denartments of *Cell Biolom and &Immunology, Duke University Medical Center, Durham, North Carolina 27710

, . -“

The mechanochemical motor proteins of the kinesin and cytoplasmic dynein families play important roles in microtubule-based intracellular motility. Although movement and distribution of organelles like secretory granules, vesicles, endoplasmic reticulum, and chromo- somes depend on the activity of these motor proteins, little is known about the regulation of this movement. We report here that the hyperphosphorylation of com- ponents of the kinesin complex by treatment with oka- daic acid increases kinesin motor activity at least 2-fold. The stimulation was observed using both a granule mo- tility assay and a microtubule gliding assay, indicating that phosphorylation enhances the activity of the motor itself, rather than the affinity of the motor for mem- brane organelles. Under stimulatory conditions, three proteins that co-purify with kinesin (with mobilities of 150, 79, and 73 kDa) are consistently hyperphosphoryl- ated. Dephosphorylation of these proteins reduces kine- sin activity to basal levels. Therefore, we conclude that kinesin motor activity is directly modulated by the phos- phorylation state of kinesin-associated proteins.

Kinesin, first identified by Vale and co-workers (1) as a plus- end-directed cytoplasmic motor, hydrolyzes ATP to produce uni- directional movement along microtubules (2-8). The rod-like kinesin (composed of two heavy chains and two light chains) has distinct globular domains (9-11). The amino-terminal end of each heavy chain forms a large globular head that contains the ATP and microtubule binding regions (10, 12-15). The smaller globular carboxyl-terminal of each chain interacts with kinesin light chains, forming a fan-shaped tail which is the putative vesicle binding domain (11, 16-18). While the struc- tural aspects of kinesin are relatively well understood, little is known about the mechanism of its regulation. Both the microtubule/ATP binding domain and the carboxyl-terminal “cargo” binding domain of kinesin are likely targets for regu- lation of motor activity. Accumulating evidence suggests that phosphorylation plays a key role in regulation of microtubule- based motors. Kinesin heavy chain (KHC)’ is phosphorylated in vivo exclusively on serine residues outside of the motor domain

*This work was supported in part by grants from the American Cancer Society (to Y. A.) and National Institutes of Health Grant GM36277 (to M. P. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Postdoctoral Fellow of the Irvington Institute for Medical Research.

Present address: Cell Biology Program, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69012 Heidelberg, Germany.

Pharmacy, 1985 Zonal Ave., Los Angeles, CA 90033. 1 1 Present address: Dept. of Pharmaceutical Sciences, USC School of

The abbreviations used are: KHC, kinesin heavy chain; KLC, kine- sin light chain; Tc, cytotoxic T lymphocytes; CEF, chicken embryo fibro- blasts; Th, T helper hybridoma; OKA, okadaic acid; PIPES, 1,4-pipera- zinediethanesulfonic acid; AMP-PNP, adenosine B’-(P,y-imino)-

(19). Kinesin light chain (KLC) is also phosphorylated at mul- tiple serine residues (19). Since the phosphorylation of the heavy and light chains occurs at the opposite end of the mol- ecule from the motor domain, this phosphorylation has been postulated to regulate kinesin binding to cargo organelles (20). In intact cells, pharmacological treatments that targeted intra- cellular phosphatases and kinases resulted in increased vesicle motility (21). Under serum-starved conditions, treatment of CV-1 cells with the serinekhreonine phosphatase inhibitor oka- daic acid increased vesicle motility by over 6-fold (21). Although these studies and others are highly suggestive, it has so far been difficult to link the observed changes in vesicle motility in vivo with changes in the phosphorylation state of microtubule motor proteins.

Two recent reports have attempted to link kinesin phospho- rylation with alterations in kinesin activity in vitro. In both studies, neuronal kinesin was phosphorylated in vitro by puri- fied protein kinases. Using CAMP-dependent protein kinase to phosphorylate highly purified kinesin, Sato-Yoshitake et al. (20) found that phosphorylated kinesin has a reduced ability to sup- port the binding of synaptic vesicles to microtubules. They con- cluded that phosphorylation of neuronal kinesin plays an es- sential role in regulating the direction of fast axonal transport, possibly by inhibiting kinesin binding to synaptic vesicles at the nerve terminals. Matthies et al. (22) also found that both CAMP- dependent protein kinase and protein kinase C can phospho- rylate kinesin in vitro. CAMP-protein kinase, which phospho- rylated KLC in preference to KHC, resulted in increased kinesin ATPase activity. These studies with highly purified components indicate that kinesin activity can be modulated by phosphoryl- ation. However, it is difficult to interpret these studies in terms of motor regulation in vivo, since pharmacological studies in intact neurons indicate that most of the known broad spectrum kinases are unlikely to phosphorylate kinesin in vivo (19).

We have taken a different approach to investigating the ef- fects of phosphorylation on organelle motility, using the phos- phatase inhibitor okadaic acid to drive motor proteins toward a more highly phosphorylated state. We investigated the role of phosphorylation in regulating motility of granules from cyto- toxic T cells, since we had previously found that they preferen- tially utilized kinesin for microtubule-dependent movement (2). Here we report that okadaic acid treatment of the cytosolic extract used to support in vitro motility increases kinesin ac- tivity. Furthermore, we identify three kinesin-associated pro- teins whose state of phosphorylation correlates with kinesin activity.

EXPERIMENTAL PROCEDURES Cell Culture-Cytotoxic T lymphocytes (Tc) were cultured as previ-

ously described (2). Briefly, precursor cells were positively selected from

triphosphate; PAGE, polyacrylamide gel electrophoresis; SUK4, mono- clonal anti-kinesin antibody.

19176

Regulation of Kinesin Motor Activity by Phosphorylation 19177

mouse splenocytes by panning for CD8 + cells and stimulated poly- clonally with 1 pg/ml concanavalin A (Aldrich). Cells were cultured in RPMI 1640 (Sigma) containing 20 unitdm1 recombinant human inter- leukin 2 (DuPont NEN), 10% rat concanavalin Asupernatant, 10% fetal calf serum, nonessential amino acids, penicillin, streptomycin (JRH Biosciences), and P-mercaptoethanol (Kodak).

Primary cultures of chicken embryo fibroblasts (CEF) were prepared from 11-day embryos as described previously (23) and maintained in Earle's minimal essential medium, supplemented with 5% fetal calf serum, penicillin, and streptomycin (all from Life Technologies, Inc.). After 2 days, primary cultures were frozen in 30% fetal calf serum, 10% dimethyl sulfoxide, 60% Iscove's modified Dulbecco's modified medium (Life Technologies, Inc.) and maintained under liquid nitrogen. Second- ary cultures were prepared from the frozen cells and grown in the supplemented Earle's minimal essential medium as described above. Secondaries were used within 2-3 days after plating.

Cultures of the murine T helper hybridoma (Th) cell line 2B23-18 (24) were maintained in Dulbecco's modified Eagle's medium high glu- cose containing 5% fetal calf serum, penicillin, and streptomycin (all from Life Technologies, Inc.).

Lytic Granule Preparation-Lytic granules were prepared as previ- ously described (2). Briefly, Tc were homogenized in disruption buffer (0.25% sucrose, 4 l ~ l ~ EGTA, 10 r m HEPES, pH 7.4) containing protease inhibitors. Nuclei and unbroken cells were removed by centrifugation at 1000 x g. The lysate was overlaid on a 48% (v/v) isotonic Percoll (Phar- macia) gradient and centrifuged at 60,000 x g for 30 min at 4 "C using a Beckman 70Ti rotor. The dense granule fractions were pooled, diluted with disruption buffer, and concentrated by centrifugation for 45 min a t 85,000 x g in a Beckman 70Ti rotor. The granule band was collected and concentrated again by centrifugation for 30 min at 100,000 x g, 4 "C, using a TLA-45 rotor (Beckman).

Preparation of Cytosol from Chick Embryo Fibroblasts and T Helper Cells-Cytosolic fractions were prepared as previously described (25, 26). For CEF cytosol, approximately 4 x 10' secondary CEF cells were collected by trypsinization. For Th cytosol, 2-3 liters of suspension culture were collected by pelleting at 1500 x g for 15 min at 4 "C. In both cases, the pelleted cells were washed with PMEE (35 mM PIPES, pH 7.4, 5 mM MgSO,, 5 mM EGTA, 0.5 mM EDTA, 1 IIM~ dithiothreitol) and protease inhibitors (26), resuspended in an equal volume of ice-cold PMEE, and homogenized using a ball bearing homogenizer. Nuclei and unbroken cells were pelleted a t 1000 x g, and the supernatant was centrifuged a t 100,000 x g for 30 min, 4 "C, in a TLS-55 rotor (Beck- man). The resulting supernatant was adjusted to 1 mM GTP (Sigma) and 20 w taxol (Calbiochem) and incubated for 15 min at 37 "C to polymerize the endogenous microtubules. The assembled microtubules were removed by centrifugation at 100,000 x g for 5 min in a Beckman Airfuge (for small volumes) or for 15 min using a TLA-45 or 100.3 rotor, (Beckman) for larger volumes. The final supernatant (S3 cytosol) was then used as the source of cytosolic motors.

Deatment of Cytosol with Okadaic Acid-S3 cytosol was made 125 I ~ M with okadaic acid (OKA, Life Technologies, Inc.) or mock-treated with an equivalent volume of PMEE and incubated for 10 min at 37 "C. If the samples were to be radiolabeled, [y3'P1ATP (Amersham, 3000 Ci/mmol) was added to 10 pCi/ml. The treated cytosol was kept on ice for use in motility assays or was used as the source for purification of motor proteins by microtubule affinity or immunoprecipitation.

Purification of Microtubule Motors-The treated S3 cytosol was ad- justed to 0.5 mg/ml phosphocellulose-purified porcine brain tubulin, 1 mM GTP, and 20 w taxol and incubated at 37 "C for 20 min to po- lymerize tubulin. The S3 cytosol-microtubule mixture was then made 2 mM AMP-PNP, 10 mg/ml glucose (both from Sigma), and 2 unitdm1 hexokinase (Boehringer Mannheim), incubated for 20 min at 25 "C, overlaid on a 25% sucrose cushion in PMEE containing 2 mM AMP-PNP, 1 mM GTP, 20 PM taxol, and centrifuged for 30 min a t 100,000 x g (Beckman TLA-45 or 100.3 depending on volume) at 25 "C. The pellet was resuspended in PMEE, 1 mM GTP, 20 p~ taxol, to one-fifth the starting volume of cytosol, and centrifuged for 15 min at 100,000 x g (25 "C). The washed pellet was then resuspended in MgATP release buffer (PMEE containing 10 mM ATP, 10 mM MgSO,, 2 mM GTP, 20 w taxol) to one-tenth the original cytosolic volume. After incubation for 15 min at 25 "C, microtubules were pelleted at 100,000 x g for 15 min (25 "C). The supernatant (MgATP eluate) was collected and kept on ice or further purified by velocity sedimentation. MgATP eluates were kept on ice for motility assays or diluted immediately in SDS sample buffer for SDS-PAGE.

Motility Assays-Granule motility assays were performed as previ- ously described (2). Porcine brain tubulin was purified by phosphocel- lulose chromatography (27) and stored at -80 "C until use. Microtu-

bules were polymerized by diluting 4 volumes of 3-3.5 mg/ml tubulin with 1 volume of PMEE containing 5 mM GTP and 100 p~ taxol and incubating at 37 "C for 15-min CTL granule assays were prepared by spotting 1 pl of one-tenth dilution of microtubule stock, 3 pl of S3 cytosol, 1.2 pl of 10 mM ATP, and 1 pl of granules directly onto a glass coverslip. Movement was visualized using contrast enhanced video dif- ferential interference microscopy (28, 29). When indicated, substitu- tions were made for the S3 cytosol.

The microtubule gliding assays were performed using a flow chamber constructed of acid-washed coverslips (20 min in 20% nitric acid, rinsed 20 min with deionized distilled water, and stored in 70% ethanol) over- laid on two strips of double stick tape on a standard microscope slide. Tape strips were spaced 2-3 mm apart, forming a chamber of 5-10 p1. The chamber was filled with 1 volume of the motor fraction, incubated for 5 min a t 24 "C, and washed with 3 volumes of 150 pg/ml casein (60% (I, 40% P, from bovine milk, Sigma) in PMEE. 2 volumes of a microtu- bule-nucleotide mixture containing 20 pVml of 3.5 mg/ml polymerized tubulin (sheared by trituration to give an average microtubule length of 3-5 pm), 10 mM ATP, 150 pg/ml casein, 10 m creatine phosphate (Boehringer Mannheim) and 80 pg/ml creatine kinase (Boehringer Mannheim) in PMEE were then flowed into the chamber and incubated for 5 min before visualizing as described above. Where indicated, sub- stitutions were made for the S3 cytosol.

Microtubule gliding assays were done using limiting dilutions of the motor fractions in the presence of excess microtubules (microtubule- nucleotide mixtures described above). Due to the variation of motor activity from different preparations, the final working dilutions were determined by directly visualizing the activity and then diluting to limiting concentrations, as measured by the number of microtubules per field in the controls. Dilutions were done in 150 pg/ml casein in PMEE.

Acid Phosphatase Deatment-The MgATP eluate from OKA-treated cytosol or mock-treated cytosol either labeled with [y3'P1ATP or unla- beled for the motility assays was treated for 15 min at 4 "C with 1 unit of desalted acid phosphatase from potato (Boehringer Mannheim) di- luted into PMEE containing protease inhibitors (see above). The mock- treated samples were similarly treated with desalted buffer minus acid phosphatase.

MetaboZicLabeling-For metabolic labeling with [35Slmethionine, 5 x lo6 CEF or Th cells were collected, resuspended in methioninekysteine- free minimum essential medium (ICN), and starved for 15 min a t 37 "C. Tran35S-label (ICN) was added to give 150 pCi/ml methionine, then cells were incubated for 4 h at 37 "C. For labeling with 32P, 5 x lo6 CEF or Th cells were resuspended in PO4-free minimum essential medium (Life Technologies, Inc.), supplemented with 3% dialyzed fetal calf serum, and starved for 15 min at 37 "C. Inorganic [32Plorthophosphate at 10 mCi/ml (Amersham) was adjusted to 1 x HEPES-buffered saline, and added to 500 pCi/ml for 4 h at 37 "C. Where indicated, 500 nM OKA was added to the labeling medium during the last 15 min of the labeling period. Cells were pelleted and lysed with Nonidet P-40 lysis medium at the end of the labeling period and prepared for immunoprecipitation as previously described (30).

Zmmunodepletion of Kinesin-OKA-treated and mock-treated T helper S3 cytosol were immunodepleted by incubation with either the anti-kinesin antibody SUK4 (31) or a nonspecific mouse IgG rotating for 2 h at 4 "C. The IgGs were removed by the addition of Protein A-agarose (Schleicher and Schuell), incubated overnight with rotation (4 "C), and then pelleted by centrifugation. The motors were then purified by mi- crotubule affinity. Motility assays were performed as described above.

RESULTS

OKA Deatment of Cytosol Stimulates Cytotoxic T Lympho- cyte Granule Motility-In a previous study using cytosol from CEF cells (21, we found that cytotoxic T lymphocyte lytic gran- ules preferentially use kinesin to bind and move along micro- tubules. Since organelle movement in intact cells is stimulated by treatment with the phosphatase inhibitor okadaic acid (OKA) (211, we asked if OKA treatment would modulate the motility of lytic granules in this reconstituted system. High speed cytosolic extract (S3 cytosol) from CEF cells was incu- bated with 125 m OKA and used in a motility assay containing taxol-stabilized microtubules, ATP, and lytic granules from cy- totoxic T cells. As shown in Fig. 1, treatment with OKA in- creased the number of granules moving on microtubules by over 2-fold. A concomitant increase in granule binding to mi-

19178 Regulation of Kinesin Motor Activity by Phosphorylation CEF Th

3.5

3.0

2.5

0 5 2.0 B - ; 1.5 rn ti

1 .o

0.5

0.0 Control +OKA -Controi +OKA

FIG. 1. OKA treatment increases the number of lytic granules bound and moving on microtubules. The high speed cytosols from chick embryo fibroblasts (CEF) and murine T helper cells ( 2 % ) were treated with 125 n~ okadaic acid (Om) or mock-treated (control) and used to support the motility of purified lytic granules in uitro. The number of granules bound and moving on microtubules was determined for each cytosol. Values are means of 108 determinations from two independent experiments * S.E.

crotubules was also observed. Similar cytosolic extracts pre- pared from a mouse T helper cell line (Th) were also capable of supporting granule motility (Fig. 1) and upon OKA treatment showed increased granule movement in parallel with binding. The fraction of bound granules moving along microtubules did not change with OKAtreatment, i.e., 21% of microtubule-bound granules moved with the CEF cytosol, and 51% of bound gran- ules moved with the Th cytosol, regardless of OKA treatment. No significant change in the rate of granule translocation was observed (data not shown). Since granule binding and motility increased in parallel, the simplest interpretation is that OKA treatment stimulates the binding activity of a microtubule mo- tor protein.

Activation Is Preserved upon Isolation of Motor Proteins- OKA could stimulate the binding of lytic granules to microtu- bules either by enhancing the interaction of motor proteins with the granule membranes or by enhancing the interaction of the membrane-motor complex with microtubules. To distin- guish between these alternatives, we measured motor activity in the absence of membranes using a quantitative microtubule gliding assay. In this assay, the number of gliding microtubules was related linearly to the motor concentration (Fig. 2 A ) . Treat- ment of the S3 cytosol from Th cells with OKA increased the number of microtubules gliding per field by 1.8-fold (from 0.26 to 0.48). The same activation was observed using in place of cytosol a motor protein preparation isolated by microtubule affinity (32). S3 from Th cells was treated with OKA or mock- treated, and motors were purified by binding to microtubules and elution with MgATP. Kinesin concentration in the MgATP eluate from OKA-treated cytosol was on average only 20% greater than in the MgATP eluate from the mock-treated cy- tosol, as determined by both Western blots and Coomassie Blue staining (data not shown). Yet, as shown in Fig. 2B, motors isolated from OKA-treated cytosol supported 2.2 times as much microtubule gliding as untreated motors (from 0.38 t 0.04 to 0.84 2 0.09: average 2 S.E.). Since OKA treatment caused a 2-fold activation of both granule motility and motor activity, we conclude that OKA stimulation of motor-microtubule interac- tions is sufficient to account for the observed effects.

Additionally, to show that kinesin was the predominant mo- tor responsible for the activity as previously observed (2), we

A

V400 1/200

r

Control +OKA

C

OKA: - + + anti - Kinesin : - + +

bound and gliding. Th cytosol was OKA-treated or mock-treated, and FIG. 2. OKA treatment increases the number of microtubules

a semipurified motor fraction was prepared by microtubule binding and elution with MgATP. Limiting dilutions of MgATP eluate were adsorbed onto acid-washed glass coverslips. Sheared taxol-stabilized microtu- bules were added in the presence of ATP, and the number of microtu- bules bound per field was determined. Vlrtually all bound microtubules were gliding. Panel A, near-limiting dilutions, the number of gliding microtubules was linearly related to the motor concentration in the quantitative microtubule gliding assay. Values are the average number of microtubules per field for each treatment at the indicated dilution. Panel B, Th MgATP eluate prepared as above was adsorbed onto acid- washed coverslips a t limiting dilutions (-1.5-2 pg/ml MgATP eluate). Values are the average of 595 fields from six independent experiments 2 S.E. Panel C, kinesin was immunoprecipitated or mock-precipitated from OKA-treated or mock-treated Th cytosol using the anti-kinesin SUK4 antibody or a nonspecific murine IgG. Motility assays were per- formed a~ above on the treated fractions.

immunodepleted the OKA-treated and mock-treated cytosol us- ing the anti-kinesin antibody, SUK4 (31). Very little motor ac- tivity was observed in the MgATP eluates from kinesin-de- pleted Th cytosols (Fig. 2C) both in the presence and absence of OKA. This observation suggests that inhibition of one or more cytosolic serinekhreonine phosphatases leads to activation of kinesin motors.

OK4 Deatment Causes Phosphorylation of Several Proteins in the MgATP Eluate-The microtubule-ATP eluate is highly enriched for both kinesin and cytoplasmic dynein along with at

Regulation of Kinesin Motor Activity by Phosphorylation

CEF Th OKA: - + - +

-205

4

-116 - 97

3 - 66

- 45

FIG. 3. Several bands in the MgATP eluate are phosphorylated in the presence of OKA. The high speed cytosols of CEF and Th cells were treated with OKA or mock-treated in the presence of [y-32PlATP. After treatment, the motors were further purified by microtubule a f in- ity. The MgATP eluate was then separated on a 7.5% SDS-PAGE gel and exposed to film. In CEF cytosol, three proteins of 150, 79, and 73 kDa were hyperphosphorylated in the presence of OKA (arrows). In Th cy- tosol, OKA-induced hyperphosphorylation of the 150- and 79-kDa pro- teins was also observed. Phosphorylation of other proteins, e.g. the 116-kDa kinesin heavy chain, sometimes occurred. These did not con- sistently correlate with OKA treatment.

least two accessory factors (32). To determine if the OKA treat- ment directly affects the phosphorylation state of any of these proteins, cytosol was incubated with [Y-~~PIATP either in the presence or absence of OKA. Microtubule affinity was then used to prepare an enriched motor fraction, and the phospho- proteins were analyzed by SDS-PAGE. In the absence of OKA, no major phosphoproteins were observed in the MgATP eluate prepared from either CEF or Th cytosol (Fig. 3). In the presence of OKA, 3 major hyperphosphorylated bands were consistently observed in motor preparations from CEF cytosol, one at 150 kDa, and a pair at 79 and 73 kDa. In motors prepared from Th cells, the 150-kDa band and the 79-kDa band were hyperphos- phorylated. Occasionally, the 116-kDa kinesin heavy chain and another protein of 100 kDa were also phosphorylated (cf. Fig. 3, Th lanes). However, neither of these proteins was consistently phosphorylated, and their phosphorylation did not always cor- relate with OKA treatment. Since the 79- and 150-kDa proteins were the only proteins in the MgATP eluates consistently hy- perphosphorylated by OKA treatment, they are candidate regu- lators of motor activity. The 73-kDa protein, which was only found in the CEF MgATP eluate, may also be important.

The Phosphorylated Proteins Co-purify with Kinesin-To de- termine whether the phosphoproteins present in the MgATP eluate are indeed associated with motor proteins and to distin- guish proteins associated with cytoplasmic dynein from those associated with kinesin, the 32P-labeled MgATP eluate from Th cells was fractionated by velocity sedimentation on 5-20% su- crose gradients. The 79- and 150-kDa bands co-sedimented with kinesin in fractions 9 through 11 (Fig. 4). While the phos- phorylated 79-kDa band was concentrated in the heaviest ki- nesin fraction, the 150-kDa phosphorylated band was found in the lighter kinesin fractions (Fig. 4). The MgATP eluate from CEF cells similarly showed that all three phosphoproteins co- migrated with kinesin (data not shown). In both cases, the phosphoproteins were absent from the fractions containing cytoplasmic dynein.

As additional verification that the phosphoproteins stimu- lated by OKAare part of the kinesin motor complex rather than fortuitously co-migrating with kinesin in the density gradient,

Fraction: 1 2 3 4 5 6 7 A

205 - 116- 97 - 66 - 45-

D D B 205 - 116 - 97 - 66 - 45 -

20%

19179

8 9 10 11 12 Load

K K K

0- DHC

0- KHC

5%

velocity sedimentation. Th cytosol was labeled with [y-32PlATP and FIG. 4. The phosphorylated bands co-sediment with kinesin by

OKA as described for Fig. 3, and the MgATP eluate was subjected to velocity sedimentation on a 5-20% sucrose gradient. Panel A shows the autoradiogram from the same gradient. The 150- and 79-kDa phospho- proteins are present exclusively in the kinesin-containing fractions. Panel B shows a Coomassie Blue-stained gel of the velocity gradient fractions. As expected, cytoplasmic dynein migrated a t 20 S (fractions 3 and 4 ) while kinesin migrated a t 9 S (fractions 9-11). D = dynein, DHC = dynein heavy chain, K = kinesin, KHC = kinesin heavy chain.

we asked whether these proteins could be immunoprecipitated with the anti-kinesin heavy chain antibody SUK4. Cytosolic extracts were incubated with [y-32P]ATP in the presence or absence of OKA, and kinesin heavy chain was immunoprecipi- tated. The 73- and 79-kDa phosphoproteins co-precipitated with KHC from both Th and CEF cells (Fig. 5) . The same experiment performed on cytotoxic T cells resulted in a similar pattern of phosphoproteins a t 150 and 70-80 kDa (data not shown). Interestingly, the 150-kDa phosphoprotein observed in both CEF and Th MgATP eluates and which co-sedimented with kinesin in the sucrose gradient from both cell types (Fig. 4 and data not shown), co-immunoprecipitated with KHC from Th cell extract but not from CEF cell extract. This result was quite reproducible (see also Fig. 7B).

Therefore, two different experimental methods identify a small set of phosphoproteins which complex with kinesin heavy chain. Furthermore, the phosphorylation state of one or more of these proteins appears responsible for the observed activation of kinesin motors. We propose to name this group of proteins - Kinesin-Associated PhosphoProteins, KAPP73, KAPP79, and KAPP150.

Dephosphorylation of the W P s Reverses Motor Acti- vation-We reasoned that if OKA-induced phosphorylation of the kinesin motor complex stimulates motility, then motor ac- tivity should be diminished by phosphatase treatment. To test this hypothesis, MgATP eluates from OKA, and mock-treated cytosols were digested with potato acid phosphatase. Phospha- tase treatment reduced microtubule gliding activity of the mo- tor fraction to control levels (Fig. 6A). Phosphatase treatment of [y-32P]ATP-labeled cytosol completely dephosphorylated the kinesin-associated phosphoproteins in the OKA-treated samples (Fig. 6B). KHC, which was phosphorylated in the above experiment, was also dephosphorylated by treatment with acid phosphatase. Thus, the activation of microtubule gliding correlates with phosphorylation: gliding increases in the presence of phosphorylated kinesin-associated proteins and decreases upon their dephosphorylation. Interestingly, al-

19180 Regulation of Kinesin Motor Activity by Phosphorylation

CEF T h ow: - + - +

-180

4

-116

- 87 + “I - 74

-61

FIG. 5. The same phosphorylated proteins co-immunoprecipi- tate with kinesin from whole cell lysates. High speed cytosol from CEF and Th cells was labeled with [y-”PIATP in the presence and absence of OKA. SUK4 anti-kinesin heavy chain antibody was used to precipitate kinesin. Although there are differences in the cell-specific set of proteins isolated with kinesin by this technique as compared with the MgATP eluate applied to sucrose gradients (see Figs. 3 and 4), the same overall set of phosphoproteins is observed using both techniques.

though the phosphatase treatment reduced the activity of the control by approximately 30%, it did not completely abolish motor activity, indicating that the dephosphorylated kinesin complex has a basal level of motor activity.

The Same Proteins Are Phosphorylated in Intact Cells-Zn vitro assays of phosphorylation frequently result in aberrant phosphorylation events. Indeed, Hollenbeck has shown that phosphorylation of KLC and KHC occurs in vitro a t sites not normally phosphorylated in vivo (19). To verify that the phos- phoproteins that we detect in cell lysates are actually phospho- rylated in intact cells, we metabolically labeled intact cells with [32P]orthophosphate. For comparison, cells were labeled in par- allel with [35Slmethionine. Cells were lysed on ice, and the kinesin motor complex was immunoprecipitated with SUK4. As shown in Fig. 7A, numerous ’?S-labeled proteins co-precipi- tated with the kinesin heavy and light chains. 32P labeling (Fig. 7B) shows that only a small number of these proteins are phosphorylated. Importantly, the pattern of kinesin-associated phosphoproteins is virtually identical with that which was ob- served in motor fractions phosphorylated in vitro (compare Fig. 7B with Fig. 5). The components of the kinesin complex could be easily metabolically labeled with 32P in intact cells, even without treatment with OKA. In contrast, phosphorylation of these proteins was not detected in the S3 cytosol unless OKA was added (cf. Fig. 3). Treatment of intact cells with up to 1 PM OKA did not increase phosphorylation of these proteins (data not shown), perhaps because their level of phosphorylation was already high. These results show that the phosphorylation de- tected in vitro has physiological relevance for motor protein regulation in vivo.

DISCUSSION

Circumstantial evidence has been accruing that phospho- rylation plays a role in the regulation of microtubule-based motors (19-20, 33, 34). KHC, KLC, and subunits of axonemal dynein have all been shown to be phosphoproteins (19, 20, 22, 35). Pharmacological treatments of intact cells that induce changes in the phosphorylation of many cellular proteins have been shown to alter organelle movement (21, 36). Phospho- rylation has never been shown, however, to directly affect mo- tor function of kinesin or cytoplasmic dynein. In this study, we demonstrate that the phosphorylation of one or more of three

A 3*50 1 T

3.00 - 2.50 - E

P 2.00 - t

2 1.50 - ln - 3 L 3 1.00 - .- E

0.50 - 0.00 -

OKA AcP:

B 205 -

116-

97 - 66 -

+ + + +

c

ow: - AcP: +

+ + - +

FIG. 6. The effects of OKA are reversed by acid phosphatase treatment. Panel A, MgATP eluates were prepared from OKA-treated or mock-treated Th cytosols, digested with potato acid phosphatase, and tested in the microtubule gliding assay described for Fig. 2. Phospha- tase treatment reversed the stimulation of gliding caused by OKA and reduced the gliding activity of untreated motors by 30%. Panel B, cy- tosol was prepared from Th cells and labeled with [y-”PIATP in the presence and absence of OKA. MgATP eluate was prepared and di- gested with potato acid phosphatase (AcP). Acid phosphatase removed all label from the kinesin heavy chain and all of the associated proteins.

kinesin-associated proteins (KAF’Ps) correlates with a 2-fold increase in kinesin motor activity. This study is the first to directly tie phosphorylation of microtubule motor-associated proteins with increased motor activity.

Using two different quantitative functional assays, we clearly demonstrate increased motor activity upon treatment of cytosol with the phosphatase inhibitor okadaic acid. The sim- plest interpretation of our results is that OKA treatment in- creases both lytic granule motility and microtubule gliding by stimulating motor-microtubule interaction, since okadaic acid treatment results in increased gliding of microtubules by motor proteins adsorbed to a glass coverslip (i.e. in the absence of organelles). In this context, it is important to note that the increase in granule motility is not an indirect result of in- creased binding of microtubules to the coverslip, since the num- ber of microtubules per field was constant (see also Ref. 2). Our results suggest that motor-microtubule interactions, rather than motor-membrane interactions, are limiting for granule motility in vitro. This finding is consistent with other data indicating that motor proteins bind to organelle membranes prior to binding to microtubules (37h2

OKA treatment results in the phosphorylation of three kine- sin-associated proteins which we have termed KAF’P73,

J. K. Burkhardt, unpublished results.

Regulation of Kinesin Motor Activity by Phosphorylation 19181

A CEF Th CEF Th OKA: - + - + OKA: - + - +

, 8. _I.

-180 m Y . '. *: - 180 .. .

""-116 - 116

.PC "87 - 87

-74 C" - 74

-61 - 61

=S 32 P FIG. 7. The kinesin-associated phosphoproteins (KAPPs) co-

immunoprecipitate with kinesin from cells metabolically la- beled with ["Plorthophosphate. CEF and Th cells were metaboli- cally labeled with [""Slmethionine (panel A ) or V'Plorthophosphate (panel B ) . Detergent lysates were prepared and kinesin heavy chain was immunoprecipitated with SUK4. Kinesin and co-precipitating pro- teins were analyzed by SDS-PAGE and autoradiography. The same phosphoproteins which co-precipitate with kinesin from [Y-~~PIATP-I~- beled cytosol are phosphorylated in vivo (compare with Fig. 5). Note that OKA treatment did not alter the degree of phosphorylation i n uiuo. Comparison with 35SS-labeled samples (panel A ) shows that the 70-80- kDa proteins are in relatively low abundance relative to kinesin heavy chain.

KAPP79, and KAPP150. Since okadaic acid is a potent inhibi- tor of PP1 and PP2A phosphatases, the KAPP proteins are probably phosphorylated on serine andor threonine residues. Consistent with this, we find that phosphorylation of these proteins is inhibited in vivo and in vitro by staurosporin, a broad-spectrum inhibitor of Sermhr kinases (data not shown).

Two of these phosphoproteins, KAPP79 and KAPP150, were consistently present in an enriched motor preparation isolated by microtubule binding and MgATP elution. Since this motor preparation exhibited all of the enhanced motor activity seen in total cytosol, these two proteins are candidates for kinesin ac- tivating factors. The correlation between KAPP phosphoryla- tion state and kinesin activity is confirmed by the reversal of stimulation of motor activity by dephosphorylation with acid phosphatase. Although KAPP150 and KAPP79 can be sepa- rated by velocity sedimentation on sucrose gradients, it has been difficult to determine whether one or both of these pro- teins shows stimulatory activity due to differences in kinesin content of the gradient fractions.

The existence of a complex containing kinesin and one or more regulatory proteins has been postulated as a requirement for the reconstitution of vesicle motility in the presence of highly purified motors (2, 32). Schroer et al. (321, in fact, iden- tified two groups of accessory factors for microtubule-based motors, dynactin and activator 11. Dynactin is a complex asso- ciated with cytoplasmic dynein, the best characterized compo- nent of which is a 150-kDa protein. Although the size of KAPPl50 is the same as that of dynactin, the purification prop- erties of the two factors on sucrose gradients are clearly dis- tinct. Activator 11, which co-purifies with kinesin, has been less well characterized. There are several proteins in the 150-kDa range of the activator I1 complex, one of which may be KAPP150.

Kinectin, a putative membrane receptor for kinesin, is a 160-kDa kinesin-binding phosphoprotein (19, 38). Kinectin is likely to be distinct from KAPP150, however, since it is a mem- brane protein which co-precipitates with kinesin only upon de- tergent lysis. In contrast, KAPPl50 co-purifies with kinesin without detergent solubilization, and we have observed no ad-

ditional KAPPl50 liberated by detergent solubilization. In ad- dition, KAPP150 does not react with a monoclonal anti-kinec- tin antibody (data not shown).

Another interesting and currently unresolved question re- garding KAPP150 is why this protein, which is present in ki- nesin gradient fractions from two T cell lines as well as CEF cells, co-immunoprecipitates with kinesin only in the two T cell lines. This difference may point to important conformational changes in KAPP150 which affect the nature of its interaction with kinesin.

Both KAPP73 and KAPP79 reproducibly co-precipitate with kinesin in all three cells lines tested, and KAPP79 also co- purifies with kinesin by microtubule affinity and sucrose gra- dient centrifugation in all three cell lines. The behavior of the smaller KAPP is more variable. It has a slightly larger molecu- lar weight in cytotoxic T lymphocytes than in Th and CEF cells and is not present in microtubule affinity preparations from Th cells. These proteins are almost certainly the same as those previously found to co-precipitate with kinesin from phosphate- labeled chick neurons (19). Interestingly, KAPP73 and KAPP79 migrate with the denser kinesin fractions in sucrose gradients. This behavior resembles that of kinesin light chain, which is found primarily in the denser kinesin fraction. One possibility is that KAPP73 and KAPP79 represent KLC isoforms, since kinesin light chains range from 60-80 kDa in various species (18). We think that this is unlikely for three reasons. First, Coomassie Blue staining and labeling with [35S]methionine re- veal that these proteins are present at very low levels relative to kinesin heavy chain ((19) and see Figs. 4B and 7). Second, these proteins fail to react with available monoclonal KLC an- tibodies. Third, the phosphopeptides generated by partial pro- teolytic digests of these proteins do not match those generated from bona fide KLC3 Nonetheless, until antibodies are avail- able that recognize conserved KLC peptides, or until the 70- 80-kDa KAPPs are isolated and partial sequences are obtained, we do not rule out the possibility that these proteins are kine- sin light chain variants.

OKA treatment has been shown to increase vesicle move- ment in serum-deprived cells (21). At least part of this stimu- lation may be due to KAPP phosphorylation. Although we find no evidence that OKA treatment leads to hyperphosphorylation of KAPP proteins in intact cells, these studies were performed in the presence of serum, where vesicle motility is already high. In keeping with this interpretation, we find that the KAPP proteins are strongly phosphorylated in cells grown in the pres- ence of serum without stimulation by OKA. I t will be interest- ing to analyze the effects of serum deprivation on KAPP phos- phorylation in vivo. It is important to note that although treatment of cytosol with OKA dramatically stimulates phos- phorylation of KAPP proteins, it has no reproducible effect on kinesin heavy chain or kinesin light chain, both of which are phosphorylated on serine residues in intact cells (19). This sug- gests that the effects of OKA on vesicle motility in vivo may be due to changes in the phosphorylation state of kinesin-associ- ated proteins rather than changes in the phosphorylation of kinesin itself.

Our results, taken together with those of Hollenbeck (191, suggest that kinesin-mediated motility is regulated at multiple levels. Based on the fact that phosphorylation of kinesin occurs on heavy chain in a small 5-kDa region near the carboxyl ter- minus and on the kinesin light chains far from the domain where ATP hydrolysis and microtubule binding take place, Hol- lenbeck (19) has proposed that phosphorylation of the kinesin chains regulates the interaction of the motor with organelle

P. J. Hollenbeck, personal communication

19182 Regulation of Kinesin Motor Activity by Phosphorylation

membranes. We show here that phosphorylation of kinesin- associated proteins affects the interaction of kinesin with mi- crotubules. Thus, phosphorylation could regulate kinesin activ- ity in two independent ways: phosphorylation of KHC and KJX could regulate binding of kinesin to organelles, while phospho- rylation of one or more of the KAPP proteins could modulate kinesin-microtubule binding. Elucidating the mechanism of motor regulation will require isolation of the individual com- ponents of the kinesin protein complex. Studies are in progress to investigate the functional role of the individual KAPP pro- teins and the nature of the phosphatase and the kinase respon- sible for the regulation of kinesin activity.

Acknowledgments-We thank J. Harris who provided valuable assis- tance with video analysis and L. Lindesmith and A. Bremer along with other members of the Sheetz and Argon Laboratories for helpful advice and criticism.

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