Regulation of kinesin-directed movements

The spatial and temporal distribution of organelles within the cell results from their transport along microtubules by motor proteins such as kinesin. This distribution is thus likely to be achieved, in part, by regulation of kinesin activity. However, ten years after the discovery of kinesinlJ, and despite the remarkable progress that has been made in the characterization of kinesin and its highly diverse family members3s4, we still do not understand how its activity is regulated. Kinesin’s ATPase activity is stimulated markedly by its interaction with microtubule9, which ensures that the motor hydrolyses significant amounts of ATP only when it can generate motility. This property, however, is not sufficient to dictate when kinesin can drive transport and when it cannot. Like myosin and dynein, kinesin can be phosphorylated6-8, and its behaviour may be modified as a consequence. We consider here how changes in phosphorylation of conventional kinesin, of a kinesin-like protein and of kinesin-associated proteins might affect transport. Kinesin

of regulation

Two models could explain regulation of bidirectional organelle transport. In one, kinesin is bound constantly to the organelle but only activated kinesin can interact with microtubules, and activation results in transport towards the microtubule’s plus end. Once kinesin is inactivated, dynein drives the organelle in the opposite direction (Fig. la). In the second model, kinesin binds transiently to organelles. When kinesin is bound, it transports the organelle towards the plus ends of microtubules. When it dissociates, dynein drives the organelle to the minus ends (Fig. lb). Cytoplasmic dynein may also be regulatedz5, and so the pathway that controls transport along microtubules may be more complex than a single on-off switch on one motor. The above models need not be mutually exclusive. Regulation could affect kinesin’s ability to interact both with the organelle and with the microtubule. TRENDS

IN CELL BIOLOGY

Bidirectional organelle transport along microtubules is most likely mediated by the opposing forces generated by two microtubule-based

VOL.

5 APRIL 1995

motors: kinesin and cytoplasmic dynein.

Because the direction and timing of organelle movements are controlled by the cell, the activity of one or both of these motor molecules must be regulated. Recent studies demonstrate that kinesin, kinesin-like proteins and kinesin-associated proteins can be phosphorylated,

function

While various members of the kinesin superfamily have been implicated in a variety of motile events, conventional kinesin appears to function primarily in organelle transport. This kinesin, composed of two identical heavy chains (KHCs) of -1 lo-130 kDa and two identical light chains (KLCs) of -60-70 kDa, was isolated on the basis of its ability to induce microtubule gliding in vitro2. Kinesin forms a rigor-like complex between microtubules and organelles in the presence of the non-hydrolysable ATP analogue AMP-PNP9, is detected on membranous vesicles by biochemical and immunofluorescence methods1°-13, promotes vesicle movements along microtubules in vitrolp19, and is required for organelle transport in vivozo,21.Because organelles move in both directions along microtubules22,23, and because many organelles, such as synaptic vesicles, bind both kinesin and cytoplasmic dynein12J4, regulation of kinesin and/or of dynein may be required to ensure that a given organelle travels along a microtubule in the appropriate direction at the appropriate time. Models

Regulation of kinesin-directed movements

phosphorylation

and suggest that changes in their state may modulate kinesin’s ability to interact

with either microtubules or organelles. Thus, it is possible that phosphorylation

regulates kinesin-driven movements.

At present, there is some evidence for each mode16,7,26,27 but not enough to favour one or exclude the other. In each case, phosphorylation has been implicated as the regulatory signal. Kinesin

phosphorylation

Both KHC and KLC can be phosphorylated in vivo7s8.In addition, kinectin, a 160 kDa integral membrane protein proposed to be a membrane receptor for kinesin28, is also phosphorylated in vivo8. All three polypeptides are modified on serine residues8 and changes in the phosphorylation of any one might affect kinesin’s behaviour. In cultures of embryonic rat hippocampal pyramidal neurons, phosphorylation of KHC and KLC is enhanced almost sevenfold and fivefold, respectively, when the cells are incubated in a membrane-permeant CAMP analogue to activate CAMP-dependent protein kinase (PKA). Furthermore, incubation of the cultures in a phorbol ester to activate protein kinase C results in an approximately threefold and twofold increase in phosphate incorporation into KHC and KLC, respectively7. Similar treatment of cultures of chick neurons, however, results in no increase in the level of phosphorylation of kinesin or kinectin, but an elevation of intracellular levels of Ca2+ in these cells decreases the phosphorylation of KLC8. These studies demonstrate that kinesin’s phosphorylation state can be altered in vivo, but they do not provide evidence as to whether a change in kinesin phosphorylation is required to control a kinesin-driven motile event. Although in vivo data are currently lacking, in vitro studies show a correlation between changes in kinesin phosphorylation and motility. 0 1995 Elsevier Science Ltd

Leah Haimo is at the Dept of Biology, University of California, Riverside, CA 92521, USA.

165

(a)

+

4

(b)

+

P FIGURE

1

Two models in which changes in the phosphorylation state of kinesin can the direction of organelle transport. (a) Kinesin motor activity is activated by phosphorylation, which enhances kinesin’s ability to interact with microtubules and results in net plus-end-directed transport of the organelle. Phosphorylation may be directly on kinesin’ or on a kinesin-associated phosphoprotein26. Dynein drives net minus-end-directed transport when kinesin is inactivated. Kinesin remains associated with the organelle during both directions of transport. (b) Kinesin binds to organelles only when it is dephosphorylated. Once bound, the kinesin drives plus-end-directed transport. Phosphorylation of kinesin induces its dissociation from the organelle and dynein can then drive minus-end-directed transpo&. As drawn, the models show regulation occurring only on kinesin, with dynein present on the organelles and active regardless of the direction of transport. Thus, active kinesin must be capable of outdriving active dynein. It is also possible that dynein, as well as

regulate

kinesin,

is regulated so that only one motor at a time. 'P' indicates phosphorylation

Phosphorylation

is active or attached to the organelle of kinesin or a kinesin-associated phosphoprotein.

affects

kinesin’s

motor

activity

Phosphorylation of kinesin, or of a protein that interacts with kinesin, might control kinesin’s ability to produce force. Purified bovine brain kinesin can be phosphorylated in vitro by a number of kinases. PKA, in particular, induces a dramatic increase in KLC phosphorylation. KLCs also bind calmodulin, as demonstrated by gel overlay methods, and calmodulin reduces the microtubule-stimulated ATPase activity of kinesin by half. Phosphorylation of kinesin by PKA reverses this calmodulin-induced 166

inhibition of ATPase activity7. Whether this phosphorylation also stimulates motility remains to be demonstrated. A recent study suggests that phosphorylation of one or more proteins that interact with kinesin, rather than of kinesin itself, may regulate its motor activityz6. Treatment of a cytosol fraction from cytotoxic T cells with okadaic acid, a protein phosphatase inhibitor, stimulates by about twofold the number of microtubules that glide and the number of T-cell lytic granules that bind to and move along microtubules in vitroz6. In addition, okadaic acid enhances vesicle transport in vivoig. Phosphorylation of three polypeptides, of 150 kDa, 78 kDa and 73 kDa, occurs when either the cytosol fraction or live cells are incubated with okadaic acid. These proteins copurify with kinesin but are present in substoichiometric amounts, so are unlikely to be kinesin subunits. They have thus been named ‘klnesin-associated phosphoproteins’26. Because these proteins are present substoichiometrically, they may activate only a small fraction of the total kinesin. To understand how phosphorylation might regulate kinesin activity, it will be necessary to map the phosphorylation sites on kinesin and/or to elucidate how the kinesin-associated phosphoproteins interact with kinesin. The kinesin motor domain, which can be excised as a 45 kDa fragment from the rest of the heavy chain by limited proteolysis, retains the ability to induce microtubule gliding in vitro3o but does not contain phosphorylated residues*. The light chains, which are phosphorylated, are located at the stalk-tail domain of KHC31. Despite the lack of phosphorylation sites in the head domain itself, there may still be a direct effect of phosphorylation on kinesin motor activity. The kinesin molecule can fold, bringing its light chains closer to its motor domain32. It is interesting that such folding inhibits klnesin’s ATPaseactivity32, and that the 45 kDa motor domain fragment or the KHC alone has higher ATPase activity than does the intact molecule30,33. These observations are consistent with KLC inhibiting motor activity. Perhaps phosphorylation affects kinesin folding and thereby kinesin’s motor activity. If phosphorylation of the kinesin-associated phosphoproteins is essential for the regulation of kinesin activity, it will be important to determine how these proteins bind to kinesin and whether this interaction is altered when they are phosphorylated. In the studies discussed above, the enhancement of kinesin activity by phosphorylation was about twofold7J6. We do not know if this level of stimulation is sufficient to regulate movements. In the presence of both kinesin and dynein, microtubules in vitro glide stochastically a few micrometres in one direction and then the other34. If this were the case in cells, then net, directed movement of organelles would not occur. It will be interesting to test whether phosphorylation of kinesin or of the kinesin-associated phosphoproteins permits kinesin to ‘out-drive’ dynein in a microtubule-gliding assay. A recent study suggests that phosphorylation of cytoplasmic dynein correlates with its dissociation from lysosomesz5, so, theoretically, a signalling event could lead to TRENDS

IN CELL BIOLOGY

VOL.

5 APRIL 1995

phosphorylation of both motors, resulting in kinesin activation and dynein dissociation from the organelle. If so, there would be no need for kinesin to overcome dynein force generation. However, as discussed below, other studies suggest that dynein remains attached to organelles during both directions of transport35, while kinesin dissociates from organelles upon its phosphorylation6. Phosphorylation organelle-binding

regulates kinesin’s properties

Kinesin is present in cells both in a soluble form and associated with organelles’i, and its distribution between the cytosol and organelles may be regulated. Immunolocalization of klnesin in ligated peripheral nerve of rat has indicated that kinesin dissociates from organellesz7. Vesicles accumulate at the plus ends of the microtubules on the proximal side of the ligation and at the minus ends of microtubules on the distal side, and kinesin immunofluorescent staining is more intense on the proximal side. Similarly, quantitative immunoelectronmicroscopy shows a 50-fold enrichment of kinesin staining on or near membranous organelles on the proximal sidez7. These studies suggest that kinesin is associated primarily with organelles that are moving towards the plus ends of the microtubules in the direction in which kinesin exerts force. Similar studies indicate that cytoplasmic dynein remains associated with organelles on both sides of a ligation35. Together these findings suggest that the association of kinesin, but not of dynein, with organelles may be regulated. Presumably, an organelle with both dynein and kinesin would move towards the plus ends of microtubules, while one with only dynein would move towards the minus ends. If so, then kinesin must outdrive dynein when both motors are present during plus-end-directed transport. The ability of kinesin to bind to organelles may be affected by its phosphorylation state. Synaptic vesicles bind about twice as much unphosphorylated kinesin as they do kinesin phosphorylated in vitro by PKA6. However, this decrease in membrane binding by phosphorylated kinesin is less than might be predicted from the in situ immunolocalization studiesz7 discussed above and may indicate that in vitro phosphorylation of kinesin by PKA does not mimic the mechanism used in vivo to regulate kinesin’s membrane association. The membrane-binding domain of kinesin is probably located in the tail domain of the molecule: bacterially expressed stalk-tail domain, but not the stalk domain alone, binds to membranes with the same affinity as native kinesin and competes with native kinesin for binding36. It will be of considerable interest to determine whether the membrane-binding properties of such kinesin fragments are affected by phosphorylation. Kinesin may unfold when it binds to membranes and thereby be released from the inhibition of its ATPaseactivity caused by folding32. Phosphorylation might simply dictate whether kinesin is bound to membranes or is soluble, while activation of the motor may be a direct consequence of unfolding. TRENDS

IN CELL BIOLOGY

VOL.

5 APRIL 1995

Regulation

of a kinesin-like

protein

Kinesin-like proteins (KLPs)possessmotor domains that share extensive sequence homology with conventional kinesin and are involved both in mitotic and meiotic movements and in organelle transport3n4. Recent studies suggest that CENP-E, a KLP implicated in mitosis, may be regulated by phosphorylation37. CENP-E accumulates in the late S/G2 phase of the cell cycle, appears at the kinetochores of chromosomes during prometaphase, relocates to the midzone during or following anaphase, and is subsequently destroyed as the cell re-enters interphase38,3g.CENP-E is phosphorylated in vivo and in vitro by maturation promoting factor (MPF)37. When phosphorylated, CENP-E can interact with microtubules only via its motor domain. When dephosphorylated, CENP-E can interact with microtubules via both its motor domain and its C-terminus, thereby crossbridging microtubules37. During prometaphase and metaphase, when MPF activity is high, CENP-E should be phosphorylated, which would mask its microtubule-crosslinking activity. Phosphorylated CENP-E, located at the kinetochores, may couple chromosomes to dynamic microtubules, and thus might be involved in prometaphase and anaphase chromosome movements40. As MPF activity falls during anaphase, CENP-E should become dephosphorylated, thus unmasking the high-affinity microtubule-binding site at its C-terminus37. This dephosphorylated motor, with two microtubulebinding sites, could then crosslink microtubules, and it may do so by dissociating from the chromosomes and relocating to the midzone during anaphase38,3g where it might be involved in generating spindle elongation or in stabilizing the overlap zone. It is interesting to consider the possibility that this KLP may have two distinct motor functions during mitosis and that it is a phosphorylation event that regulates the binding site and thus which CENP-E motor function is displayed. Which

way from

here?

The studies described here provide intriguing data suggesting that phosphorylation may modulate kinesin-driven movements and that motility may be regulated by controlling kinesin-microtubule interactions or kinesin-organelle interactions. Both interactions could be regulated if phosphorylation of kinesin at one site or phosphorylation of kinesinassociated phosphoproteins activates kinesin, while phosphorylation at a different site induces kinesin dissociation from a membrane. Similarly, phosphorylation of KLPs may dictate their binding sites and thereby regulate the movements that are driven by these motors. Thus, by controlling the phosphorylation state of these motors, the cell may regulate transport. It has not been demonstrated, however, that phosphorylation or dephosphorylation of kinesin, KLPs or kinesin-associated proteins is required for a particular intracellular motile event. As yet, genetics and molecular biology have not been applied to the study of kinesin regulation. The deletion of phosphorylation sites from these proteins should provide information concerning their 167

importance in regulating kinesin-driven intracellular motility.

21 22

References

Acknowledgements I am very grateful to David Demer for help in preparing Fig. 1 and to Bruce Telzer for critical reading of the manuscript. My work is supported by a grant from the NSF.

168

1 BRADY, S. T. (1985) Nature 317, 73-75 2 VALE, R. D., REESE, T. 5. and SHEETZ, M. P. (1985) Cell42, 39-50 3 SKOUFIAS, D. A. and SCHOLEY, 1. M. (1993) Cur-r. Opin. Cell Biol. 5, 95-l 04 4 WALKER, R. A. and SHEETZ, M. P. (1993) Annu. Rev. Biochem. 62,429-451 5 KUTZNETSOV, S. A. and CELFAND, V. I. (1986) Proc. NatlAcad. Sci. USA 83,8530-8534 6 SATO-YOSHITAKE, R., YORIFUJI, H., INAGAKI, M. and HIROKAWA, N. (1992) /. Biol. Chem. 267,23930-23936 7 MATTHIES, H. J. C., MILLER, R. J. and PALFREY, H. C. (1993) 1. Biol. Chem. 268,11176-l 1187 8 HOLLENBECK, P. J. (1993) 1~ Neurochem. 60, 2265-2275 9 LASEK, R. 1. and BRADY, S. T. (1985) Nature 316, 645-647 10 PFISTER, K. K., WAGNER, M. C., STENOIEN, D. L., BRADY, S. T. and BLOOM, C. S. (1989) 1. Ce//Bio/. 108, 1453-1463 11 HOLLENBECK, P. 1. (1989) 1. Cell Biol. 108, 2335-2342 12 LEOPOLD, P. L., McDOWALL, A. W., PFISTER, K. K., BLOOM, C. 5. and BRADY, S. T. (1992) Cell Motil. Cytoskel. 23, 19-33 13 YU, H., TOYOSHIMA, I., STEUER, E. R. and SHEETZ, M. P. (1992) /. Biol. Chem. 267,20457-20464 14 VALE, R. D., SCHNAPP, B. I., REESE, T. S. and SHEETZ, M. P. (1985) Cell 40,559-569 15 SCHROER, T. A., SCHNAPP, B. I., REESE, T. S. and SHEETZ, M. P. (1988) 1. Cell Biol. 107, 1785-l 792 16 URRUTIA, R., McNIVEN, M. A., ALBANESI, 1. P., MURPHY, D. B. and KACHAR, B. (1991) Proc. Nat/ Acad. Sci. USA 88, 6701-6705 17 BOMSEL, M., PARTON, R., KUZNETZOV, 5. A., SCHROER, T. A. and GRUENBERG, J. (1990) Cell 62,719-731 18 SCHNAPP, B. I., REESE, T. S. and BECHTOLD, R. (1992) 1. Cell Biol. 119, 389-399 19 BURKARDT, 1. K., MclLVAIN, J. K., SHEETZ, M. P. and ARGON, Y. (1993) 1. Cell Sci. 104, 151-l 62 20 HOLLENBECK, P. J. and SWANSON, J. A. (1990) Nature 346, 864-866

23 24 25 26

27

28 29 30

31 32 33 34 35 36 37 38 39 40

RODIONOV, V. I., GYOEVA, F. K. and GELFAND, V. I. (1991) Proc. Nat/ Acad. Sci. USA 88,4956-4960 SCHNAPP, B. J., VALE, R. D., SHEETZ, M. P. and REESE, T. S. (1985) Cell 40, 455-462 KOONCE, M. P. and SCHLIWA, M. (1985) 1. Cell Biol. 100, 322-326 LACEY, M. L. and HAIMO, L. T. (1992)j. Biol. Chem. 264, 47934798 LIN, S. X. H., FERRO, K. L. and COLLINS, C. A. (1994) /. Cell Biol. 127, 1009-l 019 MclLVAIN, 1. M., BURKHARDT, 1. K., HAMM-ALVAREZ, S., ARGON, Y. and SHEETZ, M. P. (1994) 1. Biol. Chem. 269, 19176-l 9182 HIROKAWA, N., SATO-YOSHITAKE, R., KOBAYASHI, N., PFISTER, K. K., BLOOM, G. S. and BRADY, 5. T. (1991) 1. Cell Viol. 114, 295-302 TOYOSHIMA, I., YU, H., STEUER, E. R. and SHEETZ, M. P. (1992) 1. Cell Biol. 118, 1121-1131 HAMM-ALVAREZ, S. A., KIM, P. Y. and SHEETZ, M. P. (1993) 1. Cell Sci. 106, 955-966 KUZNETSOV, 5. A., VAISBERG, Y. A., ROTHWELL, S. W., MURPHY, D. B. and GELFAND, V. I, (1989) 1. Biol. Chem. 264, 589-595 HIROKAWA, N., PFISTER, K. K., YORIFUJI, H., WAGNER, M. C., BRADY, S. T. and BLOOM, G. S. (1989) Cell 56, 867-878 HACKNEY, D. D., LEVITT, 1. D. and SUHAN, J. (1992) 1. Bio/. Chem. 267, 8696-8701 HACKNEY, D. D., LEVITT, J. D. and WAGNER, D. D. (1991) Biochem. Biophys. Res. Comm. 174, 81 O-81 5 VALE, R. D., MALIK, F. and BROWN, D. (1992) 1. Cell Biol. 119, 1589-l 596 HIROKAWA, N., SATO-YOSHITAKE, R., YOSHIDA, T. and KAWASHIMA, T. (1990) /, Cell Go/. 111, 1027-l 037 SKOUFIAS, D.A., COLE, D. G., WEDAMAN, K. P. and SCHOLEY, J. M. (1994) 1. Biol. Chem. 269, 1477-1485 LIAO, H., GANG, L. and YEN, T. 1. (1994) Science 265, 394-398 YEN, T. J., LI, G., SCHAAR, B. T., SZILAK, I. and CLEVELAND, D. W. (1992) Nature 359,536-539 BROWN, K. D., COULSON, R. M. R., YEN, T. 1. and CLEVELAND, D. W. (1994) /, Cell Biol. 125, 1303-l 312 LOMBILLO, V. A., NISLOW, C., YEN, T. I., GELFAND, V. I. and MCINTOSH, 1. R. (1995) /, Cell Viol. 128, 107-I 15

TRENDS

IN CELL BIOLOGY

VOL.

5 APRIL 1995