Professor of Biochemistry
Remsen Hall, Room 117
Office: 718 997-4133
FAX: 718 997-5531
Protein kinase C (PKC) is a prominent target for design of anti-cancer chemotherapeutic drugs. The occurrence of PKC as several isoforms that are structurally-related and sometimes functionally distinct complicates strategies for targeting a single isoform. This laboratory is focused on identifying intracellular substrates of specific PKC isoforms for the purpose of defining the signaling enzymatic components of isoform-stimulated pathways, and consequently to use these components as targets of chemotherapeutic agents. Targeting of a PKC isoform and one or more of its downstream targets will result in blockade of the pathway for that isoform.
I. Protein kinase C (PKC): A family of related isoforms.
PKC consists of a family of structurally-related, differentially-expressed and regulated isoforms. This suggests a complex signaling network resulting from each isoform’s ability to interact with its unique substrates while overlapping with substrates of other PKC isoforms. The PKC family of ten isoforms consists of three sub-classes, namely the diacylglycerol (DAG)-activated Ca2+-driven conventional isoforms (a, bI/bII, g), the DAG-activated Ca2+-independent novel isoforms (d, e, h, q), and the DAG-unresponsive atypical isoforms (l/i and z). Isoform-specific functions are evident in the metastasis of breast cancer cells which has been linked specifically to PKC-a, -b1, and -d, while others (e) apparently suppress this phenomenon. Inhibition of PKCa by a synthetic peptide recently was shown to decrease cell surface levels of the chemokine CXCL12 and its receptor CXCR4 of mammary tumor cells, and to inhibit metastatic activity (migration, intravasation, and matrix metalloproteinase-9 activity) in an orthotopic mouse model. Development of pharmacological agents against certain PKC isoforms and their substrates offers a promising avenue for chemotherapeutic control of breast cancer metastasis.
Engineered over-expression of PKC activity causes a profound impact on cancer-related phenotypes in non-transformed human breast cells (MCF-10A cells) such as motility, proliferation, cell morphology (Sun and Rotenberg, 1999). However, the intracellular substrates that convey the PKC signal to produce these phenotypes are largely unknown. Once a protein substrate is discovered, the functional consequences of its phosphorylated form are determined with cell-based assays that in turn link a substrate with a PKC-specific phenotype. This approach informs strategies for design of anti-cancer drugs. Based on what is known about the few PKC substrates identified to date, they tend to be cytoskeletal proteins whose phosphorylation triggers dynamic changes in the cytoskeleton that drive cell adhesion and migration. In light of the ability of overexpressed PKCa to produce aggressive movement of breast cancer cells, it continues to be an attractive anti-cancer target. However, in the majority of stage III/IV breast tumors, PKCa is down-regulated, therefore implicating a role for one or more additional PKC isoforms with the same substrate specificity as PKCa(Kerfoot et al., 2004). It is possible therefore that certain PKC isoforms converge on a few shared substrates that consequently promote events that drive cell motility and metastasis.
II. Identification of novel PKC substrates by the Traceable Kinase Method.
The Traceable Kinase Method, developed by Dr. K. Shokat (Shah et al., 1997), has been applied to several important protein kinases. This method entails construction of a PKC isoform mutant that, due to a mutation in the ATP binding site, can bind a sterically hindered ATP analogue. The FLAG-tagged mutant is expressed in non-transformed human breast cells (MCF-10A cells) and is immunoprecipitated by anti-FLAG along with any high affinity substrates. This laboratory prepared traceable kinases for PKCa, -d, and –z for the purpose of comparing their phospho-protein profiles and to identify new PKC substrates (Chen et al., in press). The findings indicate that PKCa and –d have similar phospho-protein profiles that are radically different from that of PKC-z (Abeyweera et al., 2009; Chen et al., 2012).
III. a-Tubulin is a newly identified PKC substrate.
Our published experiments (Abeyweera et al., 2009) showed that DAG-lactone treatment of MCF-10A cells (to stimulate endogenous DAG-sensitive PKC isoforms) produces phosphorylation of a-tubulin at Ser-165 and activation of aggressive cell motility. Importantly, motility of DAG-activated cells and highly metastatic human breast cells can be blocked by expression of the phosphorylation-resistant (S165N) mutant of a6-tubulin. These results strongly imply that PKC-mediated phosphorylation of a-tubulin promotes motility and therefore metastasis of human breast cancer cells.
Confocal microscopy was used to perform imaging of live cells expressing GFP-a-tubulin in order to assess the impact of a-tubulin phosphorylation on microtubule (MT) dynamics in human breast cells. MTs typically undergo phases of rapid growth and shrinking. Parameters that formalize this behavior include the rates and duration of these phases, as well as the number of catastrophes (sudden disassembly of MTs) and rescues (restoration of MT growth following catastrophe). Dynamicity is the net change in length over a period of time and describes whether there is overall growth or shrinkage of a MT for a defined time interval. These parameters have been determined for MCF-10A cells under basal conditions and following short-term PKC stimulation with DAG-lactone, a membrane-permeable PKC activator. A central finding is that DAG-lactone, which is known to stimulate aggressive motility of MCF-10A cells, causes dramatic stabilization of MTs due to a prolonged growth phase relative to the shortening phase. We are currently applying the same approach to evaluate the effect of a pseudo-phosphorylated mutant of a6-tubulin at Ser-165 (S165D) to determine whether it reproduces the effects on MT dynamics by DAG-lactone. This line of inquiry will establish whether phosphorylation of a-tubulin at Ser-165 is responsible for changes in MT dynamics that correlate with cell movement in vitro, and with metastasis in an animal model.
Abeyweera, T.P., and Rotenberg, S.A. (2007) Design and characterization of a traceable protein kinase C-a. Biochemistry 46, 2364-2370.
Abeyweera, T.P., Chen, X., and Rotenberg, S.A. (2009) Phosphorylation of a6-tubulin by protein kinase Ca activates motility of human breast cells. J. Biol. Chem. 284, 17648-17656.
Chen, X., Zhao, X., Abeyweera, T.P., and Rotenberg, S.A. (2012) Analysis of substrates of protein kinase C isoforms in human breast cells by the traceable kinase method. Biochemistry, 2012 (in press).
Kerfoot, C., Huang, W. and Rotenberg, S.A. (2004) Immunohistochemical analysis of advanced human breast carcinomas reveals down-regulation of protein kinase Ca. J. Histochem. Cytochem. 52, 419-422.
Shah, K., Liu, Y., Deirmengian, C., and Shokat, K. M. (1997) Engineering unnatural nucleotide specificity for Rous sarcoma virus tyrosine kinase to uniquely label its direct substrates. Proc. Natl. Acad. Sci. U.S.A. 94, 3565-3570.
Sun, X.-g., and Rotenberg, S.A. (1999) Over-expression of PKCa in MCF-10A human breast cells engenders dramatic alterations in morphology, proliferation and motility. Cell Growth Differ. 10, 343-352.
August 16, 2012