Essence of Cancer Biology: Disruption of Stress-fibers upon Malignant Transformation

Around 1974, at Cold Spring Harbor (CSH) Symposium on Tumor Viruses, Klaus Weber (and his colleagues) presented a “historical” paper on SV40-induced disruption of actin stress fibers in fibroblasts. SV40 is an oncogenic virus which carries the T-antigen that causes malignant transformation of normal cells such as fibroblasts. Using fluorescent antibodies against actin and myosin, they revealed that stress fibers, which consist of both actin filaments and myosin filaments, disappear  upon SV40-induced malignant transformation of fibroblasts (1).

This sensational finding prompted so many young biochemists or cancer scientists around the world to study the relationship between this cytoskeleton and malignant transformation. I was among these youth working at NIH, and in 1977 we discovered a new kinase that phosphorylates the heavy chain of a single-headed  myosin (myosin I) from a soil amoeba. This kinase is essential for the actin-activation of this myosin ATPase. Later this myosin I heavy chain kinase is among PAK (RAC/CDC42-activated kinase) family kinases, and both PAK1 and PAK4 are essential for the growth of malignant cells.   

Around 1983 at Max-Planck-Institute in Muenchen, we discovered another new kinase called “CAP42 kinase” in Physarum.  CAP42 is a heterodimer consisting of CAP42 (a) and CAP42 (b), and caps the “barbed” (plus) end of actin filaments, leading to a rapid de-polymerization of actin filaments at “pointed” (minus) end.  Capping by CAP42 is Ca²⁺-independent, but the phosphorylated (p) CAP42 requires Ca²⁺ for its capping. 

The phosphorylation takes place only at CAP42 (b), and is inhibited by actin and Ca²⁺. Furthermore, CAP42 (b), but not CAP42(a) binds DNase, as does actin.  In the end, CAP42 (b) was identified as actin, while CAP42 (a) is fragmin, an F-actin capping/severing protein of 42 kDa. Thus, “CAP42 kinase”  was renamed “Actin-Fragmin kinase” (AFK) or simply Physarum actin kinase (AK). Is there any actin kinase in mammalian cells? 

Yes, in 1986, a Japanese group found that actin is phosphorylated by CK1 (casein kinase 1) in vitro, and in 2002, a Greek group unvailed that PAK1 phosphorlates actin in mammalian cells. Thus, in mammals at least two distinct actin kinases (AKs), both of which are oncogenic. PAK1 phosphorylates both myosin and actin, while CK1 phosphorylates only actin so far.  Also it was confirmed a few years ago that SV40 T-antigen activates both PAK1 and CK1.   

Thus, it is most likely that these two kinases (PAK1 and CK1) are involved in the SV40-induced disruption of actin stress fibers upon the malignant transformation of fibroblasts that was reported by Klaus Weber et al. more than 4 decades ago. 

Klaus Weber (born in 1936) is a brilliant  German biochemist, and in my opinion, he would deserve a Nobel-prize in Physiology/Medicine for his development of a variety of popular cutting-edge biotechnologies such as SDS-PAGE, immunofluorescent staining of cytoskeleton, and SiRNA-based gene silencing. 

References:

1. R Pollack, M Osborn, and K Weber. Patterns of organization of actin and myosin in normal and transformed cultured cells. Proc Natl Acad Sci U S A. 1975; 72(3): 994–8.

 

In Quest of “Oncogenic” Actin Kinases (AKs): CK1 (Casein-Kinase 1) and PAK1

Back to 1982, when we worked at Max-Planck-Institute in Munich, Germany, we found a very peculiar kinase in Physarum which selectively phosphorylates a capping protein called Cap42 (1).  Cap42 is a hetero-dimer consisting of two subunits called Cap42 (a) and Cap42 (b).  Cap42 caps actin-filaments at the “barbed” end to block actin polymerization at this end, leading to a rapid depolymerization at the opposite (“pointed”) end of  filaments. Thus, Cap 42 causes a rapid shortening of actin filaments. This biological activity is very similar to that of Physarum fragmin and mammalian gelsolin, Ca²⁺-dependent capping/severing proteins. 

However, the capping activity of Cap42 per se is Ca²⁺-independent, and when Cap42 is phosphorylated by this peculiar kinase in Physarum, its capping activity becomes Ca²⁺-dependent. The phosphorylation takes place on Cap42 (b) only when it forms the complex with Cap42 (a), and the phosphorylation is inhibited by actin and Ca2+.  This kinase was originally called Cap42-kinase, but later renamed Actin-Fragmin Kinase (AF-kinase), because Cap42 (b) is indistinguishable from actin, while Cap42 (a) is indistinguishable from fragmin, by a few biochemical criteria (2).  AF-kinase is a protein of 80 kDa. 

Interestingly, around 1986, it was found that actin is phosphorylated by a mammalian kinase called CK1 (casein kinase 1) in vitro (3), and far later (in 2003) actin is phosphorylated in mammalian cells when they are treated with Calyculin A, a phosphatase inhibitor and Ca²⁺channel-blocker from a marine organism (4), strongly suggesting that the oncogenic kinase CK1 is a mammalian version of Physarum AK-kinase, although the Physarum AK-kinase per se fails to phosphorylate casein.  Since both CK1 and Calyculin A are oncogenic, it has been speculated that the phosphorylation of actin by these kinases also causes a malignant transformation of cells.  How is the p-actin (phosphorylated actin) oncogenic?  

In this context, it is of great interest to note that the phosphorylation sites of actin are around Thr 201-203 of actin, corresponding to the pointed end of actin filaments, to which DNase binds (2). Furthermore, DNase blocks the actin phosphorylation by these kinases. More interestingly, around 1988, a mutant of beta-actin was found to be "non-polymerizable" and oncogenic (5). In this mutant, Gly 244 is replaced by Asp, and this site is located on the “pointed” end of actin, very close to Thr 201-203 in 3D level, suggesting again that the extra negative charge(s) at the “pointed” end of actin could render actin molecule to be "non-polymerizable" and oncogenic. 

Thus, it would be of great interest to test if the Thr 201-203 Asp mutant of actin causes a malignant transformation of normal cells. It would also be of interest to test if a CK1 inhibitor blocks the Calyculin A-induced phosphorylation of actin in mammalian cells, and their oncogenic growth as well. Furthermore, we are very keen to see whether PAK1 (a myosin kinase) is involved in the activation of CK1 (an actin-kinase) or vice versa. 

If we could confirm that the p-actin is oncogenic, the CK1 inhibitor would be another good candidate for cancer therapy, as are CK2 inhibitors such as CX-4945 that eventually block PAK1 (6).  

Finally, to our hugely pleasant surprise, a Greek group  claimed that opioids-activated PI-3 kinase forms a complex with PAK1 in OK (opossum kidney) cells, and PAK1 phosphorylates actin, probably directly (7), although the phosphorylation site still remains to be clarified. 

References: 
1. Maruta H, Isenberg G, Schreckenbach T, Hallmann R, Risse G, Shibayama T, Hesse J. Ca2+-dependent actin-binding phosphoprotein in Physarum polycephalum. I. Ca2+/actin-dependent inhibition of its phosphorylation. J Biol Chem. 1983; 258(16):10144-50.
2. Gettemans J, De Ville Y, Vandekerckhove J, Waelkens E. Physarum actin is phosphorylated as the actin-fragmin complex at residues Thr203 and Thr202 by a specific 80 kDa kinase. EMBO J. 1992 ; 11(9):3185-91.
3. Shibayama T, Shinkawa K, Nakajo S, Nakaya K, Nakamura Y. Phosphorylation of muscle and non-muscle actins by casein kinase 1 in vitro. Biochem Int. 1986; 13(2):367-73.
4. Gu L, Zhang H, Chen Q, Chen J. Calyculin A-induced actin phosphorylation and depolymerization in renal epithelial cells. Cell Motil Cytoskeleton. 2003; 54(4):286-95.
5. Taniguchi S¹, Sagara J, Kakunaga T. Deficient polymerization in vitro of a point-mutated beta-actin expressed in a transformed human fibroblast cell line. J Biochem. 1988. 103(4):707-13.
6. Kim YB, Shin YJ, Roy A, Kim JH.　The Role of the Pleckstrin Homology Domain-Containing Protein CKIP-1 in Activation of p21-activated Kinase 1 (PAK1). J Biol Chem. 2015 Jul 9.

7. Papakonstanti EA, Stournaras C. Association of PI-3 kinase with PAK1 leads to actin phosphorylation and cytoskeletal reorganization. Mol Biol Cell. 2002; 13(8):2946-62.