Heterogeneous nuclear ribonucleoprotein (hnRNP) complexes, the structures that contain heterogeneous nuclear RNA and its associated proteins, constitute one of the most abundant components of the eukaryotic nucleus. hnRNPs appear to play important roles in the processing, and possibly also in the transport, of mRNA. hnRNP C proteins (Cl, Mr of 41,000; C2, Mr of 43,000 [by sodium dodecyl sulfate-polyacrylamide gel electrophoresis]) are among the most abundant pre-mRNA-binding proteins, and they bind tenaciously to sequences relevant to pre-mRNA processing, including the polypyrimidine stretch of introns (when it is uridine rich). C proteins are found in the nucleus during the interphase, but during mitosis they disperse throughout the cell. They have been shown previously to be phosphorylated in vivo, and they can be phosphorylated in vitro by a casein kinase type II. We have identified and partially purified at least two additional C protein kinases. One of these, termed Cs kinase, caused a distinct mobility shift of C proteins on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These phosphorylated C proteins, the Cs proteins, were the prevalent forms of C proteins during mitosis, and Cs kinase activity was also increased in extracts prepared from mitotic cells. Thus, hnRNP C proteins undergo cell cycle-dependent phosphorylation by a cell cycle-regulated protein kinase. Cs kinase activity appears to be distinct from the well-characterized mitosis-specific histone Hi kinase activity. Several additional hnRNP proteins are also phosphorylated during mitosis and are thus also potential substrates for Cs kinase. These novel phosphorylations may be important in regulating the assembly and disassembly of hnRNP complexes and in the function or cellular localization of RNA-binding proteins.RNA polymerase II transcripts, collectively termed heterogeneous nuclear RNAs (hnRNAs), associate as they are transcribed in the nuclei of eukaryotic cells with a specific subset of proteins to form heterogeneous nuclear ribonucleoprotein (hnRNP) complexes (12, 15). In human HeLa cells, these complexes contain, in addition to hnRNA, an assortment of at least 20 different abundant polypeptides (7, 30). As this association persists throughout their nuclear residency, it is assumed that the ensuing events that pre-mRNAs undergo to become functional translatable cytoplasmic mRNAs occur on hnRNPs rather than on naked RNA molecules.Of the ca. 20 major hnRNP proteins identified, the C proteins have occupied much of the focus of hnRNP research for several reasons. They are a prominent doublet (Cl [41 kDa] and C2 [43 kDa]) in HeLa cells) of nuclear acidic proteins which remain bound to the RNA at salt concentrations at which most of the other hnRNPs dissociate (3, 7). C proteins can also be readily UV-cross-linked to poly(A)-containing nuclear RNA in vivo (13,14,42). Immunofluorescence microscopy with monoclonal antibodies in a variety of vertebrate cells has shown that C proteins are localized to the nucleoplasm and are excluded fro...