Patent Publication Number: US-8541181-B2

Title: Myosin-IIA S1943 phosphorylation as a marker of tumor invasion

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application claims benefit of U.S. Provisional Application No. 61/453,336 filed Mar. 16, 2011, the contents of which are hereby incorporated by reference. 
    
    
     STATEMENT OF GOVERNMENT SUPPORT 
     This invention was made with government support under grant number CA100324 awarded by the National Cancer Institute. The government has certain rights in the invention. 
    
    
     SEQUENCE LISTING 
     The “.txt” Sequence Listing filed by EFS and which is entitled 96700 — 1802_ST25.txt, is 17 kilobytes in size and which was created on Feb. 3, 2012 is hereby incorporated by reference. 
     BACKGROUND OF THE INVENTION 
     Throughout this application various publications are referred to by number in parentheses. Full citations for these references may be found at the end of the specification. The disclosures of these publications are hereby incorporated by reference in their entirety into the subject application to more fully describe the art to which the subject invention pertains. 
     Although significant progress has been made in the molecular characterization of cancers, such as breast cancer, at present there are insufficient methodologies to predict risk for metastatic disease. Most biomarkers rely on correlating changes in overall protein levels with specific cancer-related biological processes; however, it is recognized that primary tumors, such as in breast cancer, exhibit alterations in signaling pathways that will affect post-translational modifications rather than elicit gross changes in protein expression levels. As a consequence, changes in post-translational modifications (e.g. phosphorylation) may be better predictors of disease. 
     The present invention addresses this need by providing a biomarker which is a predictor of metastasis and cancer prognosis. 
     SUMMARY OF THE INVENTION 
     A method is provided for determining if cells of a tumor in an organ or a tissue in a subject are likely to invade another organ or tissue in the subject comprising determining if a sample of the tumor, or a sample derived from the tumor, comprises a phosphorylated S1943 residue of a myosin-IIA heavy chain, wherein cells of the tumor are likely to invade another organ or tissue if the sample comprises the phosphorylated S1943 residue of a myosin-IIA heavy chain, and wherein absence of a phosphorylated S1943 residue of a myosin-IIA heavy chain does not indicate that cells of the tumor are likely to invade another organ or tissue. 
     A method is also provided for determining if a cancer is likely to metastasize comprising determining if a sample of the cancer, or a sample derived from the cancer, comprises a phosphorylated S1943 residue of a myosin-IIA heavy chain, wherein the cancer is likely to metastasize if the sample comprises the phosphorylated S1943 residue of a myosin-IIA heavy chain. 
     A kit is provided comprising a detectably-labeled antibody directed to phosphorylated S1943 residue of a myosin-IIA heavy chain, or a detectably-labeled fragment of an antibody which binds phosphorylated S1943 residue of a myosin-IIA heavy chain, and written instructions for using the detectably-labeled antibody or detectably-labeled fragment of an antibody to detect a phosphorylated S1943 residue of a myosin-IIA heavy chain in a sample. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1A-1B : 3D invasion assay. ( 1 A) Cartoon of assay. Tumor cells and macrophages are plated on the bottom of a MatTek dish and overlaid with a collagen-I gel. After 24 hours, the proportion of cells invading the collagen is determined by confocal microscopy. ( 1 B) Proportion of invasive MDA-MB-231 cells in the presence of macrophages (two independent experiments performed in duplicate). Inset: Blot showing endogenous non-muscle myosin heavy chain IIA (NMHC-IIA) (lower band) and GFP-NMHC-IIA mutant (upper band). 
         FIG. 2A-2B : Development of antibodies that specifically react with the NMHC-IIA pS1943. ( 2 A) pS1943 antibodies react specifically with a MDA-MB-231 whole cell extract and CK2-phosphorylated NMHC-IIA rods, but not with muscle myosin heavy chain IIA (NMHC-IIB) rods (data not shown), unphosphorylated NMHC-IIA rods, or PKC-phosphorylated NMHC-IIA rods. ( 2 B) Treatment with calf-intestine phosphatase (CIP), decreased the reactivity of the phospho-antibody with the MDA-MB-231 extract and NMHC-IIA CK2 phosphorylated rods. 
         FIG. 3 : Myosin-IIA heavy chain phosphorylation is detected in multiple human breast carcinoma cells lines. 
         FIG. 4 : MDA-MB-231 tumor. Arrows indicate localization of pS1943 NMHC-IIA in cells at the invasive edge of the tumor. 
         FIGS. 5A &amp; 5B . NMHC-IIA pS1943 immunohistochemistry of human breast tumor samples. ( 5 A) Intraductal carcinoma forming a central lumen. Arrows indicate intense pS1943 NMHC-IIA localization at the cell apex. ( 5 B) High grade carcinoma at the invasive margin. Individual tumor cells (T) have spread into the adjacent connective tissue. The arrow shows a tumor cell that has moved into a lymphatic vessel, which was identified by its flattened endothelial cell nucleus (En). There is intense staining of the tumor cell edge facing the lumen, with delicate processes extending into the luminal space. In addition to expression in tumor cells, the endothelium of a small blood vessel (V), a marginated inflammatory cell (I) and stromal fibroblasts (F) are positive. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     A method is provided for determining if cells of a tumor in an organ or a tissue in a subject are likely to invade another organ or tissue in the subject comprising determining if a sample of the tumor, or a sample derived from the tumor, comprises a phosphorylated S1943 residue of a myosin-IIA heavy chain, wherein cells of the tumor are likely to invade another organ or tissue if the sample comprises the phosphorylated S1943 residue of a myosin-IIA heavy chain, and wherein absence of a phosphorylated S1943 residue of a myosin-IIA heavy chain does not indicate that cells of the tumor are likely to invade another organ or tissue. 
     A method is provided for determining if a cancer is likely to metastasize comprising determining if a sample of the cancer, or a sample derived from the cancer, comprises a phosphorylated S1943 residue of a myosin-IIA heavy chain, wherein the cancer is likely to metastasize if the sample comprises the phosphorylated S1943 residue of a myosin-IIA heavy chain. 
     In an embodiment of the methods, determining if the sample of the tumor, or the sample derived from the tumor, or the sample of the cancer, or the sample derived from the cancer, comprises the phosphorylated S1943 residue of a myosin-IIA heavy chain comprises contacting the sample of the tumor, or the sample derived from the tumor, or the sample of the cancer, or the sample derived from the cancer, respectively, with a detectable agent which selectively binds to phosphorylated S1943 residue of a myosin-IIA heavy chain and detecting any bound detectable agent. 
     In an embodiment of the methods the tumor is a breast cancer tumor. In an embodiment of the methods the rumor is a tumor of the prostate, lung, liver, pancreas, kidney, ovary, testicle, utenis, glia, central nervous system, oesphagus, stomach, colon, or is a glioblastoma. In an embodiment of the methods, the tumor is a solid state tumor. In an embodiment, the tumor is a tumor of a solid tissue. 
     In an embodiment of the methods the detectable agent is an antibody or an antigen-binding fragment of an antibody. In an embodiment of the methods the antibody or the antigen-binding fragment of an antibody is labeled with a detectable label. In an embodiment, the detectable agent binds to the cell surface of a cell of the sample. In an embodiment, the sample is not a lysate. 
     In an embodiment of the methods the myosin-IIA is non-muscle myosin-IIA heavy chain. 
     In an embodiment of the methods the myosin-IIA heavy chain comprises consecutive amino acid residues having the sequence set forth in SEQ ID NO:1— 
     
       
         
           
               
               
            
               
                 MAQQAADKYL YVDKNFINNP LAQADWAAKK LVWVPSDKSG FEPASLKEEV GEEAIVELVE 
                   
               
               
                   
               
               
                 NGKKVKVNKD DIQKMNPPKF SKVEDMAELT CLNEASVLHN LKERYYSGLI YTYSGLFCVV 
               
               
                   
               
               
                 INPYKNLPIY SEEIVEMYKG KKRHEMPPHI YAITDTAYRS MMQDREDQSI LCTGESGAGK 
               
               
                   
               
               
                 TENTKKVIQY LAYVASSHKS KKDQGELERQ LLQANPILEA FGNAKTVKND NSSRFGKFIR 
               
               
                   
               
               
                 INFDVNGYIV GANIETYLLE KSRAIRQAKE ERTFHIFYYL LSGAGEHLKT DLLLEPYNKY 
               
               
                   
               
               
                 RFLSNGHVTI PGQQDKDMFQ ETMEAMRIMG IPEEEQMGLL RVISGVLQLG NIVFKKERNT 
               
               
                   
               
               
                 DQASMPDNTA AQKVSHLLGI NVTDFTRGIL TPRIKVGRDY VQKAQTKEQA DFAIEALAKA 
               
               
                   
               
               
                 TYERMFRWLV LRINKALDKT KRQGASFIGI LDIAGFEIFD LNSFEQLCIN YTNEKLQQLF 
               
               
                   
               
               
                 NHTMFILEQE EYQREGIEWN FIDFGLDLQP CIDLIEKPAG PPGILALLDE ECWFPKATDK 
               
               
                   
               
               
                 SFVEKVMQEQ GTHPKFQKPK QLKDKADFCI IHYAGKVDYK ADEWLMKNMD PLNDNIATLL 
               
               
                   
               
               
                 HQSSDKFVSE LWKDVDRIIG LDQVAGMSET ALPGAFKTRK GMFRTVGQLY KEQLAKLMAT 
               
               
                   
               
               
                 LRNTNPNFVR CIIPNHEKKA GKLDPHLVLD QLRCNGVLEG IRICRQGFPN RVVFQEFRQR 
               
               
                   
               
               
                 YEILTPNSIP KGFMDGKQAC VLMIKALELD SNLYRIGQSK VFFRAGVLAH LEEERDLKIT 
               
               
                   
               
               
                 DVIIGFQACC RGYLARKAFA KRQQQLTAMK VLQRNCAAYL KLRNWQWWRL FTKVKPLLQV 
               
               
                   
               
               
                 SRQEEEMMAK EEELVKVREK QLAAENRLTE METLQSQLMA EKLQLQEQLQ AETELCAEAE 
               
               
                   
               
               
                 ELRARLTAKK QELEEICHDL EARVEEEEER CQHLQAEKKK MQQNIQELEE QLEEEESARQ 
               
               
                   
               
               
                 KLQLEKVTTE AKLKKLEEEQ IILEDQNCKL AKEKELLEDR IAEFTTNLTE EEEKSKSLAK 
               
               
                   
               
               
                 LKNKHEAMIT DLEERLRREE KQRQELEKTR RKLEGDSTDL SDQIAELQAQ IAELKMQLAK 
               
               
                   
               
               
                 KEEELQAALA RVEEEAAQKN MALKKIRELE SQISELQEDL ESERASRNKA EKQKRDLGEE 
               
               
                   
               
               
                 LEALKTELED TLDSTAAQQE LRSKREQEVN ILKKTLEEEA KTHEAQIQEM RQKHSQAVEE 
               
               
                   
               
               
                 LAEQLEQTKR VKANLEKAKQ TLENERGELA NEVKVLLQGK GDSEHKRKKV EAQLQELQVK 
               
               
                   
               
               
                 FNEGERVRTE LADKVTKLQV ELDNVTGLLS QSDSKSSKLT KDFSALESQL QDTQELLQEE 
               
               
                   
               
               
                 NRQKLSLSTK LKQVEDEKNS FREQLEEEEE AKHNLEKQIA TLHAQVADMK KKMEDSVGCL 
               
               
                   
               
               
                 ETAEEVKRKL QKDLEGLSQR HEEKVAAYDK LEKTKTRLQQ ELDDLLVDLD HQRQSACNLE 
               
               
                   
               
               
                 KKQKKFDQLL AEEKTISAKY AEERDRAEAE AREKETKALS LARALEEAME QKAELERLNK 
               
               
                   
               
               
                 QFRTEMEDLM SSKDDVGKSV HELEKSKRAL EQQVEEMKTQ LEELEDELQA TEDAKLRLEV 
               
               
                   
               
               
                 NLQAMKAQFE RDLQGRDEQS EEKKKQLVRQ VREMEAELED ERKQRSMAVA ARKKLEMDLK 
               
               
                   
               
               
                 DLEAHIDSAN KNRDEAIKQL RKLQAQMKDC MRELDDTRAS REEILAQAKE NEKKLKSMEA 
               
               
                   
               
               
                 EMIQLQEELA AAERAKRQAQ QERDELADEI ANSSGKGALA LEEKRRLEAR IAQLEEELEE 
               
               
                   
               
               
                 EQGNTELIND RLKKANLQID QINTDLNLER SHAQKNENAR QQLERQNKEL KVKLQEMEGT 
               
               
                   
               
               
                 VKSKYKASIT ALEAKIAQLE EQLDNETKER QAACKQVRRT EKKLKDVLLQ VDDERRNAEQ 
               
               
                   
               
               
                 YKDQADKAST RLKQLKRQLE EAEEEAQRAN ASRRKLQREL EDATETADAM NREVSSLKNK 
               
               
                   
               
               
                 LRRGDLPFVV PRRMARKGAG DGSDEEVDGK ADGAEAKPAE 
               
            
           
         
       
     
     In an embodiment, the S1943 residue is the serine at residue number 1943 of SEQ ID NO:1. 
     Also provided is a method for determining if a cancer is likely to metastasize comprising determining if the cancer comprises a phosphorylated S1943 residue of a myosin-IIA heavy chain, wherein the cancer is likely to metastasize if the sample comprises the phosphorylated S1943 residue of a myosin-IIA heavy chain. In an embodiment, determining if the cancer comprises the phosphorylated S1943 residue of a myosin-IIA heavy chain comprises contacting the cancer with a detectable agent which selectively binds to phosphorylated S1943 residue of a myosin-IIA heavy chain and detecting any bound detectable agent. In an embodiment, the detectable agent is administered directly into the cancer, or is administered to the subject with the cancer. In an embodiment, the detectable agent is administered by injection or catheterization into the cancer. In an embodiment, the detectable agent is a detectably-labeled antibody directed to phosphorylated S1943 residue of a myosin-IIA heavy chain, or a detectably-labeled fragment of an antibody which binds phosphorylated S1943 residue of a myosin-IIA heavy chain. In an embodiment, the detectable label is fluorescent, is radioactive, or is radio-opaque. 
     Also provided is a kit comprising a detectably-labeled antibody directed to phosphorylated S1943 residue of a myosin-IIA heavy chain, or a detectably-labeled fragment of an antibody which binds phosphorylated S1943 residue of a myosin-IIA heavy chain, and written instructions for using the detectably-labeled antibody or detectably-labeled fragment of an antibody to detect a phosphorylated S1943 residue of a myosin-IIA heavy chain in a sample. 
     As used herein, the term “antibody” refers to complete, intact antibodies, “fragment of an antibody” refers to Fab, Fab′, F(ab) 2 , and other fragments thereof which fragments bind the antigen of interest, in this case phosphorylated S1943 of nonmuscle myosin-IIA heavy chain (Myh9). Complete, intact antibodies include, but are not limited to, monoclonal antibodies such as murine monoclonal antibodies, polyclonal antibodies, chimeric antibodies, human antibodies, and humanized antibodies. 
     Various forms of antibodies may be produced using standard recombinant DNA techniques (see 24). For example, “chimeric” antibodies may be constructed, in which the antigen binding domain from an animal antibody is linked to a human constant domain (an antibody derived initially from a nonhuman mammal in which recombinant DNA technology has been used to replace all or part of the hinge and constant regions of the heavy chain and/or the constant region of the light chain, with corresponding regions from a human immunoglobulin light chain or heavy chain) (see, e.g., 25 &amp; 26). Chimeric antibodies reduce the immunogenic responses elicited by animal antibodies when used in human clinical treatments. In addition, recombinant “humanized” antibodies may be synthesized. Humanized antibodies are antibodies initially derived from a nonhuman mammal in which recombinant DNA technology has been used to substitute some or all of the amino acids not required for antigen binding with amino acids from corresponding regions of a human immunoglobulin light or heavy chain. That is, they are chimeras comprising mostly human immunoglobulin sequences into which the regions responsible for specific antigen-binding have been inserted (see, e.g., PCT patent application WO 94/04679). Animals are immunized with the desired antigen, the corresponding antibodies are isolated and the portion of the variable region sequences, responsible for specific antigen binding are removed. The animal-derived antigen binding regions are then cloned into the appropriate position of the human antibody genes in which the antigen binding regions have been deleted. Humanized antibodies minimize the use of heterologous (inter-species) sequences in antibodies for use in human therapies, and are less likely to elicit unwanted immune responses. Primatized antibodies can be produced similarly. 
     Another embodiment of the antibodies employed in the compositions and methods of the invention is a human antibody, which can be produced in nonhuman animals, such as transgenic animals harboring one or more human immunoglobulin transgenes. Such animals may be used as a source for splenocytes for producing hybridomas, for example as is described in U.S. Pat. No. 5,569,825. 
     Antibody fragments and univalent antibodies may also be used in the methods and compositions of this invention. Univalent antibodies comprise a heavy chain/light chain dimer bound to the Fc (or stem) region of a second heavy chain. “Fab region” refers to those portions of the chains which are roughly equivalent, or analogous, to the sequences which comprise the Y branch portions of the heavy chain and to the light chain in its entirety, and which collectively (in aggregates) have been shown to exhibit antibody activity. A Fab protein includes aggregates of one heavy and one light chain (commonly known as Fab′), as well as tetramers which correspond to the two branch segments of the antibody Y, (commonly known as F(ab) 2 ), whether any of the above are covalently or non-covalently aggregated, so long as the aggregation is capable of specifically reacting with a particular antigen or antigen family. 
     As used herein, an agent that “selectively binds to phosphorylated S1943 residue of a myosin-IIA heavy chain”, or grammatical equivalent, means an agent which binds to myosin-IIA comprising a phosphorylated S1943 residue (“pS1943”) but which does not bind myosin-IIA having no phosphorylated S1943 residue. In non-limiting examples the agent is an antibody, or fragment of an antibody. In a preferred embodiment, the agent that selectively binds to phosphorylated S1943 residue of a myosin-IIA heavy chain does not bind to any other cellular component. 
     As used herein, a “cancer” is a disease state characterized by the presence in a subject of cells demonstrating abnormal uncontrolled replication. As used herein a “tumor” is a detectable malignant tumor usually presenting as a lesion or lump located in an organ or tissue in a subject, and may also be present in adjacent organs and or tissues in a subject. 
     As used herein “metastasize” means, in regard to a cancer or tumor, to spread from one organ or tissue of a subject to another non-adjacent organ or tissue of the subject. 
     As used herein, a “detectable agent” is any agent that binds to myosin-IIA heavy chain comprising a phosphorylated S1943 residue and which can be detected or observed, when bound, by methods known in the art. In non-limiting examples, the detectable agent can be an antibody or a fragment of an antibody, which is itself detectable, e.g. by a secondary antibody, or which is labeled with a detectable marker such as a radioisotope, a fluorophore, a dye etc. permitting detection of the presence of the bound agent by the appropriate machine, or optionally in the case of visually detectable agents, with the human eye. In an embodiment, the amount of detected agent can be quantified. 
     As used herein, a “sample” of a cancer or of a tumor is a portion of the cancer or of the tumor, respectively, for example as obtained by a biopsy. As used herein a “sample derived from a tumor” is a sample of the tumor or of the cancer which has been treated chemically and/or mechanically, but in such a manner not to remove any phosphorylated S1943 residue of a myosin-IIA heavy chain which might be contained therein and in such a manner not to phosphorylate an S1943 residue of a myosin-IIA heavy chain which was non-phosphorylated prior to such chemical and/or mechanical treatment. 
     As used herein “likely to” in regard to describing an occurrence means more likely to occur than not to occur. (90% or more of cancer deaths result from metastases (see 2010 Cancer Facts &amp; Figures, American Cancer Society. Cancer Facts &amp; Figures is an annual publication of the American Cancer Society, Atlanta, Ga.)). 
     All combinations of the various elements described herein are within the scope of the invention unless otherwise indicated herein or otherwise clearly contradicted by context. 
     This invention will be better understood from the Experimental Details, which follow. However, one skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative of the invention as described more fully in the claims that follow thereafter. 
     EXPERIMENTAL DETAILS 
     Introduction 
     Nonmuscle myosin-II is a hexameric complex comprised of two heavy chains (NMHC-II), two essential light chains and two regulatory light chains (1). Each heavy chain contains an N-terminal globular head containing the ATP and actin binding domains needed for motor activity, an intermediate coiled-coil domain and a C-terminal tailpiece. The S1943 phosphorylation site is located on the C-terminal tailpiece. The three NMHC-II isoforms (A, B, and C) exhibit distinct patterns of tissue and cell expression (2), have different enzymatic activities (3-5), interact with different proteins (6-8), and have unique functional roles in vivo (9-14). More importantly, myosin-II regulates and integrates multiple steps in the motility cycle of cells, including cell polarization and protrusion, and the assembly and turnover of adhesions (15, 16). In animal models, myosin-II regulatory pathways are activated in invasive breast cancer cells (17), and myosin-II activity is required for matrix deformation and breast cancer cell motility through a three-dimensional matrix (18). These observations suggest that myosin-II-based contractility is a critical component of carcinoma invasion. 
     Regulatory light chain phosphorylation is a well-established mechanism for activation of the myosin-II motor (19). However, there is emerging evidence from this laboratory, and others, that heavy chain phosphorylation, which promotes myosin-II filament disassembly (20, 21), provides another regulatory control for modifying myosin-II activity. Studies from this laboratory showed that EGF stimulation of human breast cancer cells induces rapid and reversible phosphorylation on S1943 of NMHC-IIA (22), which can be phosphorylated in vitro by CK2. Moreover, it was shown that expression of NMHC-IIA phosphomimetics (S1943E/D) enhances the EGF-stimulated chemotactic motility of breast cancer cells (22). 
     Results and Discussion 
     Using a previously-developed in vitro 3-D invasion assay that reconstitutes macrophage-dependent invasion of tumor cells into a collagen gel (see 23 for assay) it was observed that breast tumor cells expressing GFP-NMHC-IIA S1943E, a NMHC-IIA phosphomimetic, exhibited a 25% increase in invasion as compared to untransfected cells (p value &lt;0.00001) ( FIG. 1 ). The 3-D invasion assay results indicate the usefulness of phosphorylation status of NMHC-IIA S1943E as a metastatic marker and are significant finding because more-limited 2-D assay results are not reliably predictive of in vivo metastatic behavior. 
     Antibodies that recognize the myosin-IIA S1943 phosphorylation site were developed. Analyses demonstrated that the antibody recognizes a single band in a MDA-MB-231 breast tumor cell lysate, and that reactivity is reduced following snRNA-mediated knockdown of myosin-IIA and is abolished following treatment with calf intestinal phosphatase ( FIG. 2A ). In addition, the antibody recognized CK2-phosphorylated myosin-IIA rods (phosphorylation on S1943), but not PKC-phosphorylated rods (phosphorylation on S1916) or unphosphorylated myosin-IIA rods ( FIG. 2B ). 
     Myosin-IIA heavy chain phosphorylation is detected in multiple human breast carcinoma cells lines ( FIG. 3 ). Moreover, immunohistochemical analysis of MDA-MB-231 late stage orthotopic tumors demonstrated that S1943-phosphorylated myosin-IIA localizes to the invasive edge of MDA-MB-231 tumors ( FIG. 4 ). 
     Immunohistochemistry of human breast tumors with the pS1943 antibody showed that phosphorylated myosin-IIA selectively localizes to the free cell surfaces of breast cancer cells. Intense apical staining of the cells facing the lumen of a high grade intraductal carcinoma was observed in situ ( FIG. 5A ). In addition, in a lobular carcinoma exhibiting invasion of the surrounding stroma and lymphatic spread, invasive tumor cells at the tumor margin selectively expressed myosin-IIA at the cell periphery. When cells had penetrated intracellular spaces, such as the lumen of a lymphatic channel, the localization was confined to the leading edge of the cell ( FIG. 5B ). These observations are significant because they show that in actual human breast tumors, and not just in breast cancer cell lines which may or may not accurately reflect the in situ situation, myosin-IIA heavy chain phosphorylation is associated with tumor cell invasion. 
     In summary, the studies demonstrated that S1943 phosphorylation on the myosin-IIA heavy chain enhances breast tumor motility invasion, and is associated with invasive cells in human breast tumor samples. Since the regulation of acto-myosin contractility is critical for tumor cell migration and invasion, myosin-IIA S1943 phosphorylation serves as a prognostic indicator in human breast cancer. 
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