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Kendrew lecture- Post-translational regulation of cell signalling

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Posttranslational modification (PTM) is a means of increasing the complexity of the proteome, and reversible PTMs are commonly used in the transmission of signals within cells in response to external stimuli. Protein phosphorylation is involved in the majority of cellular processes and thousands of distinct phosphorylation events can be detected in a single cell type. The human kinome comprises >530 protein kinases of which 480 are typical eukaryotic protein kinases (ePKs), and the remainder are atypical protein kinases (aPKs); the majority are Ser/Thr kinases, but there are 90 Tyr kinases. In addition to Ser, Thr and Tyr, six other amino acids can be phosphorylated, including the three basic amino acids, His, Lys and Arg. Histidine phosphorylation of proteins is becoming increasingly relevant as a regulatory mechanism, and two aPKs, NME1 and NME2 , can reportedly phosphorylate histidine in target proteins. To study global histidine phosphorylation events we generated monoclonal antibodies to non-cleavable 1-pHis and 3-pHis analogues, and are using these mAbs to study histidine phosphorylation in normal and transformed cells and in metastasis. By using immobilized mAbs for affinity purification of pHis proteins from 293 cells, we have identified 800 proteins that bound selectively under denaturing conditions, and putatively contain 1-pHis (250) or 3-pHis (~160). We have begun to characterize some of these pHis proteins, and develop MS methods to pinpoint the exact sites of His phosphorylation. Immunostaining of HeLa cells and primary macrophages with anti-1-pHis mAbs revealed staining on the outside of phagocytic vesicles; immunostaining of proliferating HeLa cells with anti-3-pHis mAbs showed a striking pattern in mitotic cells, with strong staining of spindle poles (and centrosomes in interphase cells) and the midbody, suggesting that phosphorylation of His at the 3-position may regulate some aspects of the mitotic process.

Aberrant protein phosphorylation plays a role in many human diseases, and particularly in cancer. In this connection, we have studied the phenotypes of several newly identified cancer-associated protein kinase mutations, and have found, surprisingly, that mutations in DAPK3 , MLK4, and protein kinase C are all loss of function, implying that they act as tumor suppressors. The tumor microenvironment, which comprises many different stromal cells types in addition to tumor cells, is now recognized as playing a key role in the development of cancer. Pancreatic adenocarcinoma (PDAC) is one of the most deadly cancers, and PDAC has an unusually dense stromal matrix surrounding the tumor cells. To better understand the relationship between stromal cells and tumor cells in PDAC , we have investigated the crosstalk between pancreatic cancer cells and pancreatic stromal cells, known as stellate cells, by proteomic analysis of secreted proteins and of tyrosine phosphorylation events induced by secreted proteins in both cell types. We have found that LIF and IL-6 cytokines secreted by stellate stromal cells elicit JAK /STAT3 signaling in the tumor cells, and that PDGF secreted by the tumor cells in turn activates the stellate cells, thus setting up a reciprocal paracrine loop that could in principle be targeted for PDAC therapy. As a proof of principle that this is possible, using a mouse model of PDAC , we have found that administration of a neutralizing anti-LIF mAb prolongs survival when used in combination with gemcitabine.

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