Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.777886
Title: A novel interaction of the myosin light chain phosphatase with the regulatory subunits of protein kinase A in platelets
Author: Khalil, Jawad Shah
ISNI:       0000 0004 7963 6545
Awarding Body: University of Hull and University of York
Current Institution: University of Hull
Date of Award: 2018
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Abstract:
Platelet activation initiates a series of events, such as shape change, degranulation and aggregation, which results in the formation of a haemostatic plug and arrest of blood loss at the sites of vascular damage. Shape change is accompanied by remodelling of the actin cytoskeleton and formation of an acto-myosin contractile ring. This process is regulated by phosphorylation of the myosin light chain (MLC), which in turn is controlled by the relative activities of myosin light chain kinase (MLCK) and phosphatase (MLCP). MLCP is a target of both inhibitory and activatory signalling molecules. Here we focussed on the control of MLCP activation by the cyclic adenosine 3', 5' monophosphate (cAMP) signalling pathway. cAMP signalling is a major inhibitory pathway that inhibits platelet function through its effector protein kinase A (PKA). One of the downstream targets of PKA is MLCP. In vascular smooth muscle cells cAMP activates MLCP to dephosphorylate MLC and regulate cytoskeletal rearrangement. However, the relationship between cAMP signalling, MLCP and platelet function is unclear. Here we investigated the molecular and biochemical regulation of MLCP by cAMP signalling. We found that MYPT1, the targeting subunit of MLCP, is phosphorylated in platelets in response to cAMP elevating agents, suggesting that the phosphatase is a direct target for cAMP signalling. Our data suggests the possibility of at least two different splice variants of MYPT1 in platelets, but only one splice variant, possibly full length, was phosphorylated downstream of cAMP signalling. Using co-immunoprecipitation, cAMP pull-down assays and GST pull-down approaches we found that MYPT1 and PKA are part of the same complex and MYPT1 interacts with all four regulatory subunits of PKA (PKA-R) in platelets. Using a series of truncated proteins in HEK cells the interaction of MYPT1 and PKA-R was mapped to the central region of MYPT1 (aa501-706). Moreover, co-sedimentation assays with recombinant proteins confirmed the direct association of MYPT1 (aa501-706) with PKA-R. The conserved dimerisation and docking (D/D) domain of PKA-R, which facilitates interactions with A-kinase anchoring proteins (AKAP) was not required for the interaction. Consistent with this observation, the AKAP disruptor peptide Ht31 did not disrupt the interaction, indicating that the interaction of MYPT1 with PKA-R does not occur in an AKAP modus. These results were complemented with immunohistochemistry studies showing that MYPT1 co-localised with all four PKA regulatory subunits in both non-activated and spread platelets. In summary, our data identify MYPT1 as a novel PKA binding protein in platelets. The interaction of MYPT1 with PKA emerges as an important component of the signalling pathways that protect platelets from a hyperreactive state and may constitute a target towards preventing thrombogenic disorders.
Supervisor: Rivero-Crespo, Francisco ; Naseem, Khalid Sponsor: University of Hull
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.777886  DOI: Not available
Keywords: Medicine
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