Primal de Lanerolle

Myosin I and Actin in the Nucleus.
GTPase-Myosin Interactions in Cell Signalling
Vascular/Smooth Muscle Biology

Myosin I and II are members of a superfamily of actin-activated molecular motors that convert chemical energy into mechanical work. Most members of this superfamily do not form filaments.  Myosin I is the best studied of these "unconventional" myosins.  We discovered a nuclear isoform of myosin I that contains an unique 16 amino acid NH2-terminal extension.  Light and electron microscopy revealed that nuclear myosin I co-localizes with RNA polymerase I and II and biochemical and cell biology studies established that nuclear myosin I is essential for transcription by both polymerases.

Myosin molecules work in concert with actin and actin is also abundant in the nucleus. We have shown that actin is necessary for pre-initiation complex (PIC) formation, apparently because it facilitates the integration of the RNA polymerase II complex into the developing PIC. Interestingly, NMI is not needed for forming PICs, but it does appear to be necessary for forming the first few bonds during mRNA synthesis. Other experiments have shown that both actin and NMI stimulate transcription by RNA polymerase I and II.

We have also recently demonstrated that actin and nuclear myosin I are involved in chromosome movements in mammalian nuclei. The nucleus is organized into compartments and inactive or late replicating chromosomes are found near the nuclear envelope (heterochromatin in Fig. 1) while “active” chromosomes are found near the center of the nucleus. Previously, it was thought that the movement of chromosomes from one domain to the other occurred by a process called constrained diffusion. However, using wild type and mutant forms of actin and nuclear myosin I, we have shown that chromosome movement in an active process.

Figure 1: Possible roles for actin and nuclear myosin I (NMI) in the nucleus. Actin binds tightly to RNA polymerase I and II while NMI binds to DNA. Thus, NMI heads sticking out from the DNA backbone could interact with actin bound to polymerases to function as a motor during transcription. NMI bound to DNA could also bind to short actin polymers to move active genes to the nuclear interior.

Our current research focuses on investigating the regulation of actin dynamics and analyzing the proteins that actin and nuclear myosin I bind to in the nucleus. Myosin V and myosin VI have also been discovered in the nucleus and we are studying how they, too, are involved in various nuclear functions. Myosin VI is especially intriguing because it moves in the opposite direction on actin filaments compared to all other myosins and it is likely to have unique functions in the nucleus.

Myosin II is also a major research focus in my lab. Actin and myosin II are major constituents of the cytoskeleton and, by generating force, they determine the physical characteristics of a cell.  The actin-myosin II interaction in smooth muscle and non-muscle cells is regulated by the phosphorylation of ser 19 of the 20 kDa light chain of myosin II by the enzyme myosin light chain kinase (MLCK). We, and others, have shown that myosin II phosphorylation is modulated by small G proteins (GTPases), such as Rho and Ras.  GTPases regulate a host of cellular responses and we have proposed that myosin II phosphorylation is an important part of the integrated cellular response to GTPase activation.

Figure 2:  A high resolution analysis of the MLCK promoter using chloroacetaldehyde showed the formation of triplex H-DNA structures and that a mutation in SHR promoter forms a longer H-DNA structure than the promoter from normotensive rats.

We have used spontaneously hypertensive rats (SHR), a widely-used model of hypertension, to investigate this hypothesis. As with humans, blood pressure increases as SHR become older. In addition, hypertension in SHR is also highly dependent on the activation of the Ras signaling pathway by angiotensin II. We have shown that a mutation in the promoter increases MLCK expression and myosin light chain phosphorylation in SHR. We have also found that this mutation changes the structure of the MLCK promoter (Figure 2), making it more responsive to Ras signaling. Importantly, we have shown that inhibiting Ras signaling prevents the development of hypertension. We are now analyzing genomic DNA from hypertensive patients to determine if there are mutations that increase MLCK expression in human hypertension.

Selected Publications (Click here for PubMed List of Publications)

1. de Lanerolle, P. and Stull, J.T. Myosin phosphorylation during contraction and relaxation of tracheal smooth muscle. J. Biol. Chem. 255: 9993-10000, 1980.

2. de Lanerolle, P., Adelstein, R.S., Feramisco, J.R. and Burridge, K. Characterization of anti-bodies to smooth muscle myosin kinase and their use in localizing myosin kinase in non-muscle cells. Proc. Nat. Acad. Sci. USA 78: 4738-4742, 1981.

3. de Lanerolle, P., Condit, J.R., Tanenbaum, M. and Adelstein, R.S. Myosin phosphorylation, agonist concentration and contraction of tracheal smooth muscle. Nature 298: 871-872, 1982.

4. de Lanerolle, P., Nishikawa, M., Yost, D.A. and Adelstein, R.S. Increased phosphorylation of myosin light chain kinase after an increase in cyclic AMP in intact smooth muscle. Science 223: 1415-1417, 1984.

5. de Lanerolle, P. and Nishikawa, M. Regulation of fetal smooth muscle myosin by protein kinase C. J. Biol. Chem. 263: 9071-9074, 1988.

6. Wilson, A.K., Gorgas, G., Claypool, W.D. and de Lanerolle, P. An increase or a decrease in myosin II phosphorylation inhibits macrophage motility. J. Cell Biology 114: 277-283, 1991.

7. de Lanerolle, P. and Paul, R.J. Myosin phosphorylation/dephosphorylation and the regulation of airway muscle contractility. Am. J. Physiol. 258: L1-L14, 1991.

8. de Lanerolle, P., Gorgas, G., Li, X. and Schluns, K. Myosin light chain phosphorylation does not increase during yeast phagocytosis by macrophages. J. Biol. Chem. 268: 16,883-16,886, 1993.

9. Christensen, S., Verhage, H.G., Nowak, G., de Lanerolle, P., Fleming, S., Bell, S.C. Smooth muscle myosin II and alpha smooth muscle actin expression in the baboon (papio anubis) uterus is associated with glandular secretory activity and stromal cell transformation. Biology of Reproduction 53: 596-606, 1995.

10 Hecht, G.A., Pestic, L., Nikcevic, G., Koutsouris, A., Tripuraneni, J., Lorimer, D.D., Nowak, G., Guerriero, V., Elson, E. and de Lanerolle, P. Expression of the catalytic domain of myosin light chain kinase increases paracellular permeability. Am. J. Physiol. 271: C1675-C1684, 1996.

11. Klemke, R.L., Cai, S., Giannini, A.L., Gallagher, P., de Lanerolle, P. and Cheresh, D.A. Regulation of cell motility by mitogen activated protein kinase. J. Cell Biol. 137: 481-492, 1997.

12. Nowak, G., Pestic-Dragovich, L., Hozak, P., Philimonenko, A., Simerly, C., Schatten, G. and de Lanerolle, P. Evidence for the presence of myosin I in the nucleus. J. Biol. Chem. 272: 17,176-17,181, 1997.

13. Majumdar, M., Seasholtz, T.M., Goldstein, D., de Lanerolle, P. and Brown, J.H. Thrombin-induced cell rounding is mediated through Rho and requires myosin light chain phosphorylation. J. Biol. Chem. 273: 10,099-10,106, 1998.

14. Cai, S., Pestic-Dragovich, L., O'Donnell, M.E., Wang, N., Ingber, D., Elson, E. and de Lanerolle, P. Regulation of cytoskeletal dynamics and cell growth by myosin light chain phosphorylation. Am. J. Physiol. 275: C1349-C1356, 1998.

15. Sanders, L., Matsumura, F., Bokoch, G.M. and de Lanerolle, P. Inhibition of myosin light chain kinase by p21-activated kinase (PAK) during cell spreading. Science 283: 2083-2085, 1999.

16. Pestic-Dragovich, L., Stojiljkovic, L., Philimonenko, A., Nowak, G., Ke, Y., Settlage, R.E., Shabanowitz, J., Hunt, D.F., Hozak, P. and de Lanerolle, P. A myosin I isoform in the nucleus. Science 290: 337-341, 2000

17. Qu., J., Cammarano, M.S., Shi, Q, Ha, K.C., de Lanerolle, P. and Minden, A. Activated PAK 4 regulates cell adhesion and anchorage independent growth. Mol. Cell. Biol. 21: 3523-33, 2001

18. Yuan, S.Y., Wu, M.H., Ustinova, E.E., Guo, M., Tinsley, J.H., de Lanerolle, P. and Xu, W. Myosin light chain phosphorylation in neutrophil-stimulated coronary microvascular leakage. Circulation Research 90: 1214-1221, 2002.

19. de Lanerolle, P. and Cole, A.B. Cytoskeletal proteins and gene regulation: Form, function and signal transduction in the nucleus. Science's STKE, content/full/sigtrans. July 2, 2002.

20. Hofmann, W.A., Ljuba Stojiljkovic, L., Fuchsova, B., Vargas, G.M., Mavrommatis, E., Philimonenko, V., Kysela, K., Goodrich, J.A., Lessard, J.L., Hope, T.J., Hozak, P. and de Lanerolle, P. Actin is part of pre-initiation complexes and necessary for transcription by RNA polymerase II. Nature Cell Biology 6: 1094-1101, 2004.

21. Philimonenko, V.V., Zhao, J., Iben, S., Dingova, H., Kysela, K., Kahle, M., Zentgraf, H., Hofmann, W.A., de Lanerolle, P., Hozak, P. and Grummt, I. Nuclear actin and myosin I are required for RNA polymerase I transcription. Nature Cell Biology 6: 1165-1172, 2004.

22. de Lanerolle, P. Top ten tips for success in graduate school. Nature 433: 442, 2005.

23. Fazal, F., Gu, L-Z., Ihnatovych, I., Han, YJ., Antic, N., Hu, W-Y., Carriera, F., Blomquist, J.F., Hope, T.J., Ucker, D. and de Lanerolle, P. Inhibiting myosin light chain kinase induces apoptosis in vitro and in vivo. Mol. Cell. Biology 25: 6259-6266, 2005.

24. de Lanerolle, P., Johnson, T. and Hofmann, W.A. Actin and myosin I in the nucleus: What next? Nature Struct. and Molec. Biol., 12: 292-296, 2005.

25. Chuang, C-H., Carpenter, A.E., Fuchsova, B., Johnson, T., de Lanerolle, P. and Belmont, A.S. Long-range directional movement of an interphase chromosome site. Current Biology 16: 825-831, 2006.

26. Gu, L., Hu, W-Y., Antic, N., Mehta, R., Turner. J.R. and de Lanerolle, P. Inhibiting Myosin Light Chain Kinase Retards the Growth of Mammary and Prostate Cancer Cells, In Vitro and In Vivo. European Journal of Cancer 42: 948-957, 2006. Epub Mar 30, 2006.

27. Hofmann, W.A., Vargas, G.M., Ramchandran, R., Stojiljkovic, L., Goodrich, J.A. and de Lanerolle, P. Nuclear myosin I is necessary for the formation of the first phosphodiester bond during transcription initiation by RNA polymerase II. J. Cellular Biochemistry , 99: 1001-1009, 2006.

28. Han, Y-J., Hu, W-Y., Antic, N., Gu, L., Gupta, M., Piano, M. and de Lanerolle, P. Increased myosin light chain kinase expression in hypertension: Regulation by SRF via an insertion mutation in the promoter. Molecular Biology of the Cell 17: 4039-4050, 2006.

29. Hu, W-Y., Han, Y-J., Gu, L-Z., Piano, M. and de Lanerolle, P. Involvement of myosin light chain phosphorylation in the captopril effects in spontaneously hypertensive rats. American Journal of Hypertension : 20: 53-61, 2007.

30. Ihnatovych, I., Hu, W-Y., Martin, J.L., Fazleabas, A.T., de Lanerolle, P. and Strakova, Z. Increased phosphorylation of myosin light chain prevents human uterine fibroblast decidualization, Endocrinology , 148: 3176-3184, 2007.

31. Han, Y-J. and de Lanerolle, P . Naturally extended CT.AG repeats increase H-DNA structures and histone modifications in the smooth muscle myosin light chain kinase promoter. Mol. Cell. Biology 28: 863-872, 2008.

32 Han, Y-J., Hu, W-Y., Piano, M. and de Lanerolle, P . Regulation of myosin light chain kinase expression by angiotensin II during vascular remodeling. American Journal of Hypertension, 21: 860-865, 2008.

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