The unique N‐terminal region of SRMS regulates enzymatic activity and phosphorylation of its novel substrate docking protein 1

Abstract

SRMS (Src-related tyrosine kinase lacking C-terminal regulatory tyrosine and N-terminal myristoylation sites) belongs to a family of nonreceptor tyrosine kinases, which also includes breast tumour kinase and Fyn-related kinase. SRMS, similar to breast tumour kinase and Fyn-related kinase, harbours a Src homology 3 and Src homology 2, as well as a protein kinase domain. However, unlike breast tumour kinase and Fyn-related kinase, SRMS lacks a C-terminal regulatory tail but distinctively possesses an extended N-terminal region. Both breast tumour kinase and Fyn-related kinase play opposing roles in cell proliferation and signalling. SRMS, however, is an understudied member of this family. Although cloned in 1994, information on the biochemical, cellular and physiological roles of SRMS remains unreported. The present study is the first to explore the expression pattern of SRMS in breast cancers, its enzymatic activity and autoregulatory elements, and the characterization of docking protein 1 as its first bonafide substrate. We found that, similar to breast tumour kinase, SRMS is highly expressed in most breast cancers compared to normal mammary cell lines and tissues. We generated a series of SRMS point and deletion mutants and assessed enzymatic activity, subcellular localization and substrate recognition. We report for the first time that ectopically-expressed SRMS is constitutively active and that its N-terminal region regulates the enzymatic activity of the protein. Finally, we present evidence indicating that docking protein 1 is a direct substrate of SRMS. Our data demonstrate that, unlike members of the Src family, the enzymatic activity of SRMS is regulated by the intramolecular interactions involving the N-terminus of the enzyme and that docking protein 1 is a bona fide substrate of SRMS. Structured digital abstract• SRMS physically interacts with Dok-1 by pull down (View Interaction: 1, 2)• Dok-1 physically interacts with SRMS by anti bait coimmunoprecipitation (View Interaction: 1, 2, 3)• SRMS phosphorylates Dok-1 by protein kinase assay (View interaction)• Dok-1 physically interacts with SRMS by anti tag coimmunoprecipitation (View interaction) Abbreviations BRK, breast tumour kinase; DAB, 3,3′-diaminobenzidine tetrahydrochloride; DAPI, 4′,6-diamidino-2-phenylindole; Dok1, downstream of tyrosine kinases 1/docking protein 1; FRK, Fyn-related kinase; GFP, green fluorescent protein; GST, glutathione S-transferase; HEK 293, human embryogenic kidney 293; IHC, immunohistochemistry; PEI, polyethyleneimine; PTK, protein tyrosine kinase; PDN-SRMS, partial N-terminus deletion SRMS; SH2, Src-homology 2; SH3, Src-homology 3; SRMS, Src-related tyrosine kinase lacking C-terminal regulatory tyrosine and N-terminal myristoylation sites; DN-SRMS, N-terminus deletion SRMS.

Protein-tyrosine Phosphatase and Kinase Specificity in Regulation of SRC and Breast Tumor Kinase*

“…It was reported that SRMS is overexpressed in breast tumors, whereas its expression level in normal mammary epithelial cells is very low (41). It was also reported that SRMS phosphorylates DOK1, which has been implicated in mitogenic signaling pathways in chronic myelogenous leukemia (41). Previous studies have indicated BRK is subject to similar regulatory mechanisms as SRC (20), and we are currently characterizing the SRMS-BRK interaction in different cell contexts and subcellular localizations.…”

“…CSK is a negative regulator of kinase activity and has tumor suppressor function (15), but the cellular functions of SRMS are still unclear. It was reported that SRMS is overexpressed in breast tumors, whereas its expression level in normal mammary epithelial cells is very low (41). It was also reported that SRMS phosphorylates DOK1, which has been implicated in mitogenic signaling pathways in chronic myelogenous leukemia (41).…”

Understanding the cellular roles of Fyn-related kinase (FRK): implications in cancer biology

“…This lysine residue forms the ATP-contacting site in FRK which remains conserved in several receptor and non-receptor tyrosine kinases including SFKs and BFKs. Mutating this conserved lysine residue in SFKs as well as BRK and SRMS has been shown to result in loss of catalytic activity in these enzymes [22][23][24].…”

“…In Src family member Hck, substitution of the corresponding W260 with an alanine enhances the catalytic activity of Hck by causing a disruption in the interaction between W260 and the N-lobe of the kinase domain thereby promoting an active conformation of the enzyme [25]. Paradoxically, a similar mutagenic substitution in BRK (W184A) and SRMS (W223A) resulted in significantly suppressed catalytic potential of these enzymes [22,24]. This is perhaps due to the local conformational geometry that may differ between SFKs and BRK/SRMS.…”

mRNA expression of DOK1-6 in human breast cancer

“…Goel et al [58] have recently published qualitative data of a lucid differential pattern of DOK-1 expression in 8 breast cancer cell lines compared to a non-tumourigenic breast epithelial-derived cell line. We believe our study to be the first that has quantitatively measured DOK-1 expression in human breast cancer specimens.…”